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71.
Johannes Hirrlinger Anja Scheller Petra G. Hirrlinger Beate Kellert Wannan Tang Michael C. Wehr Sandra Goebbels Andreas Reichenbach Rolf Sprengel Moritz J. Rossner Frank Kirchhoff 《PloS one》2009,4(1)
Cre/LoxP recombination is the gold standard for conditional gene regulation in mice in vivo. However, promoters driving the expression of Cre recombinase are often active in a wide range of cell types and therefore unsuited to target more specific subsets of cells. To overcome this limitation, we designed inactive “split-Cre” fragments that regain Cre activity when overlapping co-expression is controlled by two different promoters. Using transgenic mice and virus-mediated expression of split-Cre, we show that efficient reporter gene activation is achieved in vivo. In the brain of transgenic mice, we genetically defined a subgroup of glial progenitor cells in which the Plp1- and the Gfap-promoter are simultaneously active, giving rise to both astrocytes and NG2-positive glia. Similarly, a subset of interneurons was labelled after viral transfection using Gad67- and Cck1 promoters to express split-Cre. Thus, split-Cre mediated genomic recombination constitutes a powerful spatial and temporal coincidence detector for in vivo targeting. 相似文献
72.
73.
Importance of the N-distal AP-2 binding element in Nef for simian immunodeficiency virus replication and pathogenicity in rhesus macaques
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Brenner M Münch J Schindler M Wildum S Stolte N Stahl-Hennig C Fuchs D Mätz-Rensing K Franz M Heeney J Ten Haaft P Swigut T Hrecka K Skowronski J Kirchhoff F 《Journal of virology》2006,80(9):4469-4481
Point mutations in SIVmac239 Nef disrupting CD4 downmodulation and enhancement of virion infectivity attenuate viral replication in acutely infected rhesus macaques, but changes selected later in infection fully restore Nef function (A. J. Iafrate et al., J. Virol. 74:9836-9844, 2000). To further evaluate the relevance of these Nef functions for viral persistence and disease progression, we analyzed an SIVmac239 Nef mutant containing a deletion of amino acids Q64 to N67 (delta64-67Nef). This mutation inactivates the N-distal AP-2 clathrin adaptor binding element and disrupts the abilities of Nef to downregulate CD4, CD28 and CXCR4 and to stimulate viral replication in vitro. However, it does not impair the downmodulation of CD3 and class I major histocompatibility complex (MHC-I) or MHC-II and the upregulation of the MHC-II-associated invariant chain, and it has only a moderate effect on the enhancement of virion infectivity. Replication of the delta64-67Nef variant in acutely infected macaques was intermediate between grossly nef-deleted and wild-type SIVmac239. Subsequently, three of six macaques developed moderate to high viral loads and developed disease, whereas the remaining animals efficiently controlled SIV replication and showed a more attenuated clinical course of infection. Sequence analysis revealed that the deletion in nef was not repaired in any of these animals. However, some changes that slightly enhanced the ability of Nef to downmodulate CD4 and moderately increased Nef-mediated enhancement of viral replication and infectivity in vitro were observed in macaques developing high viral loads. Our results imply that both the Nef functions that were disrupted by the delta64-67 mutation and the activities that remained intact contribute to viral pathogenicity. 相似文献
74.
Kaiser M Maletzki I Hülsmann S Holtmann B Schulz-Schaeffer W Kirchhoff F Bähr M Neusch C 《Journal of neurochemistry》2006,99(3):900-912
Transgenic mice expressing the superoxide dismutase G93A mutation (SOD1(G93A)) were used to investigate the role of glial inwardly rectifying K(+) (Kir)4.1 channels, which buffer extracellular K(+) increases in response to neuronal excitation. A progressive decrease in Kir4.1 immunoreactivity was observed predominantly in the ventral horn of SOD1(G93A) mutants. Immunoblotting of spinal cord extracts mirrored these changes by showing a loss of Kir4.1 channels from presymptomatic stages onwards. Kir4.1 channels were found to be expressed in the spinal cord grey matter, targetting astrocytes and clustering around capillaries, supporting their role in clearance of extracellular K(+). To understand the functional implications of extracellular K(+) increases, we challenged the NSC34 motor neurone cell line with increasing extracellular K(+) concentrations. Exposure to high extracellular K(+) induced progressive motor neurone cell death. We suggest that loss of Kir4.1 impairs perineural K(+) homeostasis and may contribute to motor neurone degeneration in SOD1(G93A) mutants by K(+) excitotoxic mechanisms. 相似文献
75.
Carol F. Kirchhoff Xiao‐Zhuo Michelle Wang Hugh D. Conlon Scott Anderson Anne M. Ryan Arindam Bose 《Biotechnology and bioengineering》2017,114(12):2696-2705
A biosimilar drug is defined in the US Food and Drug Administration (FDA) guidance document as a biopharmaceutical that is highly similar to an already licensed biologic product (referred to as the reference product) notwithstanding minor differences in clinically inactive components and for which there are no clinically meaningful differences in purity, potency, and safety between the two products. The development of biosimilars is a challenging, multistep process. Typically, the assessment of similarity involves comprehensive structural and functional characterization throughout the development of the biosimilar in an iterative manner and, if required by the local regulatory authority, an in vivo nonclinical evaluation, all conducted with direct comparison to the reference product. In addition, comparative clinical pharmacology studies are conducted with the reference product. The approval of biosimilars is highly regulated although varied across the globe in terms of nomenclature and the precise criteria for demonstrating similarity. Despite varied regulatory requirements, differences between the proposed biosimilar and the reference product must be supported by strong scientific evidence that these differences are not clinically meaningful. This review discusses the challenges faced by pharmaceutical companies in the development of biosimilars. 相似文献
76.
Mallikarjun S Beelagi SR Santosh Kumar Uma Bharathi Indrabalan Sharanagouda S Patil Ashwini Prasad KP Suresh Shiva Prasad Kollur Veeresh Santhebennur Jayappa Siddappa B Kakkalameli Chandrashekar Srinivasa Prabhakarareddy Anapalli Venkataravana Chandan Shivamallu 《Bioinformation》2021,17(4):479
Crimean-Congo hemorrhagic fever (CCHF) virus is one among the major zoonosis viral diseases that use the Hyalomma ticks as their transmission vector to cause viral infection to the human and mammalian community. The fatality of infectious is high across the world especially in Africa, Asia, Middle East, and Europe. This study regarding codon usage bias of S, M, and L segments of the CCHF virus pertaining to the host Homo sapiens, reveals in-depth information about the evolutionary characteristics of CCHFV. Relative Synonymous Codon Usage (RSCU), Effective number of codons (ENC) were calculated, to determine the codon usage pattern in each segment. Correlation analysis between Codon adaptation index (CAI), GRAVY (Hydrophobicity), AROMO (Aromaticity), and nucleotide composition revealed bias in the codon usage pattern. There was no strong codon bias found among any segments of the CCHF virus, indicating both the factors i.e., natural selection and mutational pressure shapes the codon usage bias. 相似文献
77.
Rajesh Abraham Jacob Cassandra R. Edgar Jrmie Prvost Steven M. Trothen Antony Lurie Mitchell J. Mumby Alexa Galbraith Frank Kirchhoff S.M. Mansour Haeryfar Andrs Finzi Jimmy D. Dikeakos 《The Journal of biological chemistry》2021,297(3)
Prolonged immune activation drives the upregulation of multiple checkpoint receptors on the surface of virus-specific T cells, inducing their exhaustion. Reversing HIV-1-induced T cell exhaustion is imperative for efficient virus clearance; however, viral mediators of checkpoint receptor upregulation remain largely unknown. The enrichment of checkpoint receptors on T cells upon HIV-1 infection severely constrains the generation of an efficient immune response. Herein, we examined the role of HIV-1 Nef in mediating the upregulation of checkpoint receptors on peripheral blood mononuclear cells. We demonstrate that the HIV-1 accessory protein Nef upregulates cell surface levels of the checkpoint receptor T-cell immunoglobulin mucin domain-3 (Tim-3) and that this is dependent on Nef''s dileucine motif LL164/165. Furthermore, we used a bimolecular fluorescence complementation assay to demonstrate that Nef and Tim-3 form a complex within cells that is abrogated upon mutation of the Nef dileucine motif. We also provide evidence that Nef moderately promotes Tim-3 shedding from the cell surface in a dileucine motif–dependent manner. Treating HIV-1-infected CD4+ T cells with a matrix metalloprotease inhibitor enhanced cell surface Tim-3 levels and reduced Tim-3 shedding. Finally, Tim-3-expressing CD4+ T cells displayed a higher propensity to release the proinflammatory cytokine interferon-gamma. Collectively, our findings uncover a novel mechanism by which HIV-1 directly increases the levels of a checkpoint receptor on the surface of infected CD4+ T cells. 相似文献
78.
Regulation of death receptor-mediated apoptosis pathways 总被引:10,自引:0,他引:10
Schmitz I Kirchhoff S Krammer PH 《The international journal of biochemistry & cell biology》2000,32(11-12):1123-1136
Apoptosis or programmed cell death can be induced by a variety of stimuli including activation of death receptors. This subgroup of the TNF/NGF-receptor-superfamily activates caspases, a family of aspartyl-specific cysteine-proteases, which are the main executioners of apoptosis. Depending on the cell type, signalling pathways downstream of the death receptors can be modulated by different proteins such as Bcl-2, FLIPs, chaperones and kinases. Deregulation of apoptosis has been associated with diseases as cancer, autoimmunity and AIDS. Therefore, the identification of modulators of apoptosis has several therapeutic implications. 相似文献
79.
Neuronal or glial progeny: regional differences in radial glia fate 总被引:27,自引:0,他引:27
Malatesta P Hack MA Hartfuss E Kettenmann H Klinkert W Kirchhoff F Götz M 《Neuron》2003,37(5):751-764
The precursor function of the ubiquitous glial cell type in the developing central nervous system (CNS), the radial glia, is largely unknown. Using Cre/loxP in vivo fate mapping studies, we found that radial glia generate virtually all cortical projection neurons but not the interneurons originating in the ventral telencephalon. In contrast to the cerebral cortex, few neurons in the basal ganglia originate from radial glia, and in vitro lineage analysis revealed intrinsic differences in the potential of radial glia from the dorsal and ventral telencephalon. This shows that the progeny of radial glia not only differs profoundly between brain regions but also includes the majority of neurons in some parts of the CNS. 相似文献
80.
Nadia R. Roan Haichuan Liu Shariq M. Usmani Jason Neidleman Janis A. Müller Aram Avila-Herrera Ali Gawanbacht Onofrio Zirafi Simon Chu Ming Dong Senthil T. Kumar James F. Smith Katherine S. Pollard Marcus F?ndrich Frank Kirchhoff Jan Münch H. Ewa Witkowska Warner C. Greene 《Journal of virology》2014,88(13):7221-7234