Genome editing with targeted nucleases and DNA donor templates homologous to the break site has proven challenging in human hematopoietic stem and progenitor cells (HSPCs), and particularly in the most primitive, long-term repopulating cell population. Here we report that combining electroporation of zinc finger nuclease (ZFN) mRNA with donor template delivery by adeno-associated virus (AAV) serotype 6 vectors directs efficient genome editing in HSPCs, achieving site-specific insertion of a GFP cassette at the CCR5 and AAVS1 loci in mobilized peripheral blood CD34 HSPCs at mean frequencies of 17% and 26%, respectively, and in fetal liver HSPCs at 19% and 43%, respectively. Notably, this approach modified the CD34CD133CD90 cell population, a minor component of CD34 cells that contains long-term repopulating hematopoietic stem cells (HSCs).
View Article and Find Full Text PDFIn polarized T cells, HIV-1 Gag localizes to a rear-end protrusion known as the uropod in a multimerization-dependent manner. Gag-laden uropods participate in formation of virological synapses, intercellular contact structures that play a key role in cell-to-cell HIV-1 transmission. Our previous observations suggest that Gag associates with uropod-directed microdomains (UDMs) that eventually comigrate with Gag to the uropod over the cell surface.
View Article and Find Full Text PDFTetherin/BST-2 (here called tetherin) is an antiviral protein that restricts release of diverse enveloped viruses from infected cells through physically tethering virus envelope and host plasma membrane. For HIV-1, specific recruitment of tetherin to assembly sites has been observed as its colocalization with the viral structural protein Gag or its accumulation in virus particles. Because of its broad range of targets, we hypothesized that tetherin is recruited through conserved features shared among various enveloped viruses, such as lipid raft association, membrane curvature, or ESCRT dependence.
View Article and Find Full Text PDFHIV-1 particle assembly is driven by the structural protein Gag. Gag binds to and multimerizes on the inner leaflet of the plasma membrane, eventually resulting in formation of spherical particles. During virus spread among T cells, Gag accumulates to the plasma membrane domain that, together with target cell membrane, forms a cell junction known as the virological synapse.
View Article and Find Full Text PDFProper subcellular localization of focal adhesion kinase (FAK) is crucial for many cellular processes. It remains, however, unclear how FAK activity is regulated at subcellular compartments. To visualize the FAK activity at different membrane microdomains, we develop a fluorescence resonance energy transfer (FRET)-based FAK biosensor, and target it into or outside of detergent-resistant membrane (DRM) regions at the plasma membrane.
View Article and Find Full Text PDFT cells adopt a polarized morphology in lymphoid organs, where cell-to-cell transmission of HIV-1 is likely frequent. However, despite the importance of understanding virus spread in vivo, little is known about the HIV-1 life cycle, particularly its late phase, in polarized T cells. Polarized T cells form two ends, the leading edge at the front and a protrusion called a uropod at the rear.
View Article and Find Full Text PDFThe chemoenzymatic installation of the clinically valuable (S)-4-amino-2-hydroxybutyryl side chain onto a number of 2-deoxystreptamine-containing aminoglycosides is described using the purified Bacillus circulans biosynthetic enzymes BtrH and BtrG in combination with a synthetic acyl-SNAC surrogate substrate.
View Article and Find Full Text PDFButirosin, an aminoglycoside antibiotic produced by Bacillus circulans, bears the unique (S)-4-amino-2-hydroxybutyrate (AHBA) side chain, which protects the antibiotic from several common resistance mechanisms. The AHBA side chain is advantageously incorporated into clinically valuable antibiotics such as amikacin and arbekacin by synthetic methods. Therefore, it is of significant interest to explore the biosynthetic origins of this useful moiety.
View Article and Find Full Text PDFThe proteins Neo-11 and Neo-18 encoded in the neomycin gene cluster (neo) of Streptomyces fradiae NCIMB 8233 have been characterized as glucosaminyl-6'-oxidase and 6'-oxoglucosaminyl:L-glutamate aminotransferase, respectively. The joint activity of Neo-11 and Neo-18 is responsible for the conversion of paromamine to neamine in the biosynthetic pathway of neomycin through a mechanism of FAD-dependent dehydrogenation followed by a pyridoxal-5'-phosphate-mediated transamination. Neo-18 is also shown to catalyze deamination at C-6''' of neomycin, thus suggesting bifunctional roles of the two enzymes in the formation of both neosamine rings of neomycin.
View Article and Find Full Text PDFNat Prod Rep
December 2006
The 2-deoxystreptamine-containing aminoglycosides are an important class of clinically valuable antibiotics. A deep understanding of the biosynthesis of these natural products is required to enable efforts to rationally manipulate and engineer the biological production of novel aminoglycosides. This review discusses the development of our biosynthetic knowledge over the past half-century, with emphasis on the relatively recent contributions of molecular biology to the elucidation of these biosynthetic pathways.
View Article and Find Full Text PDFMacaques were immunized with SF162 Env-based gp140 immunogens and challenged simultaneously with the CCR5-tropic homologous SHIV(SF162P4) and the CXCR4-tropic heterologous SHIV(SF33A) viruses. Both mock-immunized and immunized animals became dually infected. Prior immunization preferentially reduced the viral replication of the homologous virus during primary infection but the relative replication of the two coinfecting viruses during chronic infection was unaffected by prior immunization, despite the fact that five of six immunized animals maintained a significantly lower overall viral replication that the control animals.
View Article and Find Full Text PDFButirosins A and B are naturally occurring aminoglycoside antibiotics that have a (2S)-4-amino-2-hydroxybutyrate (AHBA) side chain. Semisynthetic addition of AHBA to clinically valuable aminoglycoside antibiotics has been shown both to improve their pharmacological properties and to prevent their deactivation by a number of aminoglycoside-modifying enzymes involved in bacterial resistance. We report here that the biosynthesis of AHBA from L-glutamate, encoded within a previously identified butirosin biosynthetic gene cluster, proceeds via intermediates tethered to a specific acyl carrier protein (ACP).
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