1,720,971 research outputs found
Synthesis of Cell-Penetrating Peptides and Their Application in Neurobiology
No full textShort basic amino acid sequences, often called cell-penetrating peptides (CPPs), allow the delivery of proteins and other molecules into cells and across the blood-brain barrier (BBB). Although the ability of basic proteins to facilitate such trafficking is known for a long time, only the application of genetic methods and overexpression of fusion proteins in Escherichia coli has lead to a wide application of CPP in many research areas, including signal transduction, cancer, angiogenesis, apoptosis, bone development, cardioprotection, cell cycle, neurobiology, and many others. For the neuroscientist, CPPs are particularly attractive, as a number of articles in the last 5 years have reported their use for neuronal rescue in a number of models for neurodegenerative diseases in vitro and in vivo in rats, mice, or gerbils. Here, we give a detailed description of the protein purification methodology and applications in neuroscience
Ginkgo biloba leaf extract EGb 761(®) as a paragon of the product by process concept
No full textIt is an often-neglected fact that extracts derived from the very same plant can differ significantly in their phytochemical composition, and thus also in their pharmacokinetic and pharmacodynamic properties which are the basis for their clinical efficacy and safety. The Ginkgo biloba L. [Ginkgoaceae] special extract EGb 761(®) is one of the best-studied plant extracts in the world. In the present review, using that extract as a paradigm, we describe insights how climate, the harvest region, processing of the plant material, the drying process, the extraction solvents, and the details of the subsequent process steps substantially impact the quality and uniformity of the final extract. We highlight the importance of regulating active constituent levels and consistent reduction of undesired substances in herbal extracts. This is accomplished by a controlled production process and corresponding analytical specifications. In conclusion, since extracts derived from the same plant can have very different phytochemical compositions, results from pharmacological, toxicological and clinical studies gained with one specific extract cannot be extrapolated to other extracts that were generated using different production processes. We propose that the heterogenous nature of extracts should be meticulously considered when evaluating the efficacy and safety of plant-derived remedies
The TAT Protein Transduction Domain Enhances the Neuroprotective Effect of Glial-Cell-Line-Derived Neurotrophic Factor after Optic Nerve Transection
No full textGlial-cell-line-derived neurotrophic factor (GDNF) acts as a potent survival factor for many neuronal populations, including retinal ganglion cells (RGC), indicating a potential therapeutic role of GDNF for neurological disorders. To enhance the tissue distribution and applicability of the neurotrophin, we linked it to a protein transduction domain derived from the HIV TAT protein and tested it in a well-established model for traumatic injury in the CNS: After optic nerve axotomy, the number of surviving RGCs was significantly increased in mice injected with TAT-GDNF on days 0, 3, 7, and 10 after surgery compared with GDNF- or PBS-injected animals. Moreover, TAT-GDNF reduced the number of activated caspase-3-positive cells. These results show that the neuroprotective effect of substances like neurotrophins may be enhanced by linking them to a domain that has been shown to mediate efficient transduction across biological membranes. Copyright (C) 2004 S. Karger AG, Base
Expression of the protein inhibitor of nitric oxide synthase in the adult rat retina before and after optic nerve lesion
No full textThe molecular messenger nitric oxide (NO) not only serves a number of physiologic functions, but is also involved in the pathophysiology of neurodegeneration. It is produced by the nitric oxide synthase (NOS) isoenzymes. One of the many players regulating NOS activity is the Protein Inhibitor of NOS, PIN. To gain further insight into the mechanisms of NOS regulation and NO-mediated cell death after nerve trauma, we examined PIN expression in a standard model of lesion-induced neurodegeneration, the rat optic nerve transsection model. In both the axotomized retinae and the control retinae PIN expression was predominantly observed in the retinal ganglion cell layer. Optic nerve lesion did neither change the amount of PIN mRNA, as determined by in situ hybridization and real-time RT-PCR, nor did it change the amount of PIN as determined by immunohistochemistry and Western blot analysis. These results suggest that in our model, NOS activity is not regulated by altered PIN levels, which contributes to our understanding of apoptotic mechanisms in injured neurons. (c) 2005 Elsevier B.V. All rights reserved
Intravenous TAT-GDNF is protective after focal cerebral ischemia in mice
No full textBackground and Purpose-Delivery of therapeutic proteins into tissues and across the blood-brain barrier is severely limited by their size and biochemical properties. The 11-amino acid human immunodeficiency virus TAT protein transduction domain is able to cross cell membranes and the blood-brain barrier, even when coupled with larger peptides. The present studies were done to evaluate whether TAT-glial line-derived neurotrophic factor (GDNF) fusion protein is protective in focal cerebral ischemia. Methods-Anesthetized male C57BL/6j mice were submitted to intraluminal thread occlusion of the middle cerebral artery. Reperfusion was initiated 30 minutes later by thread retraction. Laser Doppler flow was monitored during the experiments. TAT-GDNF, TAT-GFP (0.6 nmol each), or vehicle was intravenously applied over 10 minutes immediately after reperfusion. After 3 days (30 minutes of ischemia), animals were reanesthetized and decapitated. Brain injury was evaluated by histochemical stainings. Results-Immunocytochemical experiments confirmed the presence of TAT-GDNF protein in the brains of fusion protein-treated nonischemic control animals 3 to 4 hours after TAT fusion protein delivery. TAT-GDNF significantly reduced the number of caspase-3-immunoreactive and DNA-fragmented cells and increased the number of viable neurons in the striatum, where disseminated tissue injury was observed, compared with TAT-GFP- or vehicle-treated animals. Conclusions-Our results demonstrate that TAT fusion proteins are powerful tools for the treatment of focal ischemia when delivered both before and after an ischemic insult. This approach may be of clinical interest because such fusion proteins can be intravenously applied and reach the ischemic brain regions. This approach may therefore offer new perspectives for future strategies in stroke therapy
Inhibition of neuronal apoptosis in vitro and in vivo using TAT-Mediated protein transduction
No full textThe HIV TAT protein contains an 11-amino-acid protein transduction domain which acts as a "Trojan peptide": Linked to other macromolecules, it carries them across cellular membranes. Here, we demonstrate for the first time that fusion of the TAT protein transduction domain to an antiapoptotic protein represents a feasible technique to rescue neurons from apoptotic degeneration in vitro and in vivo. When fused to the antiapoptotic protein Bcl-X-L it mediated uptake of the fusion protein into neurons. Once inside the cells, TAT-Bcl-X-L was stable for many days and maintained its antiapoptotic function. It completely blocked low-potassium-induced apoptosis of cerebellar granule cells in vitro. In vivo, 24% of mouse retinal ganglion cells were prevented from undergoing retrograde neuronal apoptosis caused by optic nerve lesion when TAT-Bcl-X-L was intraocularly injected. The application of TAT fusion proteins may in the future greatly facilitate neuroprotective therapy strategies for neurological disorders
Delivery of bioactive molecules into the cell: the Trojan horse approach
No full textIn recent years, vast amounts of data on the mechanisms of neural deand
regeneration have accumulated. However, only in disproportionally
few cases has this led to efficient therapies for human patients. Part
of the problem is to deliver cell death-averting genes or gene products
across the blood– brain barrier (BBB) and cellular membranes. The
discovery of Antennapedia (Antp)-mediated transduction of heterologous
proteins into cells in 1992 and other ''Trojan horse peptides''
raised hopes that often-frustrating attempts to deliver proteins would
now be history. The demonstration that proteins fused to the Tat
protein transduction domain (PTD) are capable of crossing the BBB
may revolutionize molecular research and neurobiological therapy.
However, it was only recently that PTD-mediated delivery of proteins
with therapeutic potential has been achieved in models of neural
degeneration in nerve trauma and ischemia. Several groups have
published the first positive results using protein transduction domains
for the delivery of therapeutic proteins in relevant animal models of
human neurological disorders. Here, we give an extensive review of
peptide-mediated protein transduction from its early beginnings to new
advances, discuss their application, with particular focus on a critical
evaluation of the limitations of the method, as well as alternative
approaches. Besides applications in neurobiology, a large number of
reports using PTD in other systems are included as well. Because each
protein requires an individual purification scheme that yields sufficient
quantities of soluble, transducible material, the neurobiologist will
benefit from the experiences of other researchers in the growing field of
protein transduction
Peptide-enhanced cellular internalization of proteins in neuroscience
No full textOver the last 15 years, many publications described the use of peptide sequences that have been dubbed cell penetrating peptides (CPP), Trojan Horse peptides, protein transduction domains, or membrane-translocating sequences. These mostly positively charged domains bring attached cargo across biological membranes. One of the reasons for the interest in CPP is their potential as delivery tools to enhance the pharmacodynamics of drugs otherwise poorly bioavailable. In particular, the neuroscientist aiming to deliver a protein or other compound into the brain for analytical or therapeutic reasons is faced with the challenge that few drugs cross the blood-brain barrier. CPP are valuable tools to overcome the plasma membrane or the blood-brain barrier in basic research, and in relevant models of neural disease, and will hopefully help to increase the precious few treatments or even cures for people with diseases of the brain and nervous system. Here, we review applications in neuroscience and recent insights into the mechanism of CPP-mediated trafficking. (c) 2005 Elsevier Inc. All rights reserved
Stearylated octaarginine and artificial virus-like particles for transfection of siRNA into primary rat neurons
No full textRNA interference (RNAi) provides a powerful experimental tool for sequence-specific gene silencing, allowing efficient analysis of gene function in a multitude of cell types. However, application of RNAi in primary mammalian neurons has been limited by low-transfection efficiency and considerable toxicity of conventional transfection methods. In this study, we evaluated a peptide-mediated and a polymer/lipid-based cellular delivery method for siRNA into rat primary neurons and compared the results with a commonly used liposomal transfection reagent. Stearylated octaarginine (Stearyl-R8) was used as polypeptide and artificial virus-like particles (AVPs) were used as a combined liposomal-polymeric vector, since both reagents have been previously shown to successfully transfect DNA into cell lines. Stearyl-R8 and AVPs both promoted siRNA transfection into primary hippocampal neurons via the endosomal pathway. SiRNA-mediated gene silencing could be effectively induced in primary neuron cultures. In comparison with the commonly used cationic liposome transfection agent, both novel reagents were less detrimental to cell metabolic activity. We conclude that these novel transfection methods yield performances comparable to cationic liposome-mediated transfection for siRNA, while being less cytotoxic in primary neurons. Stearyl-R8 and AVPs may therefore represent novel and more cost-efficient alternatives to conventional siRNA-transfection reagents
Limited protection of TAT-Bcl-X-L against pneumolysin-induced neuronal cell death
No full textSevere brain damage in patients with pneumococcal meningitis is in part caused by the cytosolic pneumococcal protein pneumolysin. The devastating effect of this neurotoxin might be alleviated by interfering with the cell death pathways that it sets in motion. An important player in these pathways is Bcl-X(L), an antiapoptotic protein of the Bcl-2 family, which is neuroprotective in various in vitro and in vivo models of cell death. We investigated whether its membrane-permeable form, the TAT-Bcl-X(L) fusion protein, is capable of protecting human SH-SY5Y neuroblastoma cells against pneumolysin-induced cell death. Under mild pneumolysin-induced neuronal injury, TAT-Bcl-X(L) increased cell viability significantly by approximately 40% (82.7 +/- 16.1% versus 70.0+/-8.2%; p = 0.04). When the cells were exposed to a more rigorous pneumolysin treatment, TAT-Bcl-X(L) had no protective effects. This suggests the involvement of additional neuronal death pathways in pneumolysin-induced cell death, which are not controlled by Bcl-X(L). Therefore, Bcl-X(L), a promising therapeutic candidate for ischemia and neurodegenerative diseases, is only of partial efficacy in preventing the direct neurotoxicity of pneumolysin.</p
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