Dendritic cells (DCs) consist of various subsets that play crucial roles in linking innate and adaptive immunity. In the murine spleen, CD8α(+) DCs exhibit a propensity to ingest dying/dead cells, ...produce proinflammatory cytokines, and cross-present Ags to generate CD8(+) T cell responses. To track and ablate CD8α(+) DCs in vivo, we generated XCR1-venus and XCR1-DTRvenus mice, in which genes for a fluorescent protein, venus, and a fusion protein consisting of diphtheria toxin receptor and venus were knocked into the gene locus of a chemokine receptor, XCR1, which is highly expressed in CD8α(+) DCs. In both mice, venus(+) cells were detected in the majority of CD8α(+) DCs, but they were not detected in any other cells, including splenic macrophages. Venus(+)CD8α(+) DCs were superior to venus(-)CD8α(+) DCs with regard to their cytokine-producing ability in response to TLR stimuli. In other tissues, venus(+) cells were found primarily in lymph node (LN)-resident CD8α(+), LN migratory and peripheral CD103(+) DCs, which are closely related to splenic CD8α(+) DCs, although some thymic CD8α(-)CD11b(-) and LN CD103(-)CD11b(-) DCs were also venus(+). In response to dsRNAs, diphtheria toxin-treated XCR1-DTR mice showed impaired CD8(+) T cell responses, with retained cytokine and augmented CD4(+) T cell responses. Furthermore, Listeria monocytogenes infection and anti-L. monocytogenes CD8(+) T cell responses were defective in diphtheria toxin-treated XCR1-DTRvenus mice. Thus, XCR1-expressing DCs were required for dsRNA- or bacteria-induced CD8(+) T cell responses. XCR1-venus and XCR1-DTRvenus mice should be useful for elucidating the functions and behavior of XCR1-expressing DCs, including CD8α(+) and CD103(+) DCs, in lymphoid and peripheral tissues.
Chain orientation of poly(glycolic acid) (PGA) induced by halloysite nanotubes (HNTs) in hybrid electrospun fibers was investigated. The well-aligned PGA/HNTs hybrid fibers were prepared by ...electrospinning. The influence of HNTs loading (0, 1, 5, 10 wt%) on the PGA molecular chain and crystallite orientation was characterized by polarized Fourier transform infrared spectroscopy (FT-IR) and wide angle X-ray diffraction (WAXD). The effect of HNTs loading to the thermal properties of PGA fibers was investigated by differential scanning calorimetry (DSC), and the reinforcement effect of HNTs for PGA fibers was evaluated by tensile test. High temperature FT-IR measurement revealed the presence of hydrogen bonding between PGA carbonyl groups and HNTs surface silanol groups, which induces the alignment of PGA crystallite along the HNTs long axis. At low HNTs loading, HNTs distributed and aligned along the fiber axis during electrospinning process, whereas at high HNTs loading, the aggregation and anisotropic alignment of HNTs in PGA fibers lead disordered PGA chain orientation. The PGA molecular chain and crystallite orientation achieved the highest orientation degree at 5 wt% HNT loading. Furthermore, DSC results indicated that HNTs act as nucleating agent for PGA fibers and further increase the crystallinity of PGA fibers. Besides, young's modulus of PGA fibers was enhanced significantly with the addition of HNTs, which indicates a high reinforcement effect of HNTs to PGA fibers.
Bacterial polysulfide reductase (PsrABC) is an integral membrane protein complex responsible for quinone-coupled reduction of polysulfide, a process important in extreme environments such as deep-sea ...vents and hot springs. We determined the structure of polysulfide reductase from Thermus thermophilus at 2.4-A resolution, revealing how the PsrA subunit recognizes and reduces its unique polyanionic substrate. The integral membrane subunit PsrC was characterized using the natural substrate menaquinone-7 and inhibitors, providing a comprehensive representation of a quinone binding site and revealing the presence of a water-filled cavity connecting the quinone binding site on the periplasmic side to the cytoplasm. These results suggest that polysulfide reductase could be a key energy-conserving enzyme of the T. thermophilus respiratory chain, using polysulfide as the terminal electron acceptor and pumping protons across the membrane via a previously unknown mechanism.
Plasmacytoid dendritic cells (pDCs), originating from hematopoietic progenitor cells in the BM, are a unique dendritic cell subset that can produce large amounts of type I IFNs by signaling through ...the nucleic acid–sensing TLR7 and TLR9 (TLR7/9). The molecular mechanisms for pDC function and development remain largely unknown. In the present study, we focused on an Ets family transcription factor, Spi-B, that is highly expressed in pDCs. Spi-B could transactivate the type I IFN promoters in synergy with IFN regulatory factor 7 (IRF-7), which is an essential transcription factor for TLR7/9-induced type I IFN production in pDCs. Spi-B–deficient pDCs and mice showed defects in TLR7/9-induced type I IFN production. Furthermore, in Spi-B–deficient mice, BM pDCs were decreased and showed attenuated expression of a set of pDC-specific genes whereas peripheral pDCs were increased; this uneven distribution was likely because of defective retainment of mature nondividing pDCs in the BM. The expression pattern of cell-surface molecules in Spi-B–deficient mice indicated the involvement of Spi-B in pDC development. The developmental defects of pDCs in Spi-B–deficient mice were more prominent in the BM than in the peripheral lymphoid organs and were intrinsic to pDCs. We conclude that Spi-B plays critical roles in pDC function and development.
Methicillin-resistant Staphylococcus aureus (MRSA) is currently one of the principal multiple drug resistant bacterial pathogens causing serious infections, many of which are life-threatening. ...Consequently, new therapeutic targets are required to combat such infections. In the current work, we explore the type 2 Nicotinamide adenine dinucleotide reduced form (NADH) dehydrogenases (NDH-2s) as possible drug targets and look at the effects of phenothiazines, known to inhibit NDH-2 from Mycobacterium tuberculosis. NDH-2s are monotopic membrane proteins that catalyze the transfer of electrons from NADH via flavin adenine dinucleotide (FAD) to the quinone pool. They are required for maintaining the NADH/Nicotinamide adenine dinucleotide (NAD+) redox balance and contribute indirectly to the generation of proton motive force. NDH-2s are not present in mammals, but are the only form of respiratory NADH dehydrogenase in several pathogens, including S. aureus. In this work, the two putative ndh genes present in the S. aureus genome were identified, cloned and expressed, and the proteins were purified and characterized. Phenothiazines were shown to inhibit both of the S. aureus NDH-2s with half maximal inhibitory concentration (IC50) values as low as 8μM. However, evaluating the effects of phenothiazines on whole cells of S. aureus was complicated by the fact that they are also acting as uncouplers of oxidative phosphorylation. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.
•Two new type 2 NADH dehydrogenases from S. aureus are purified and characterized.•A new protocol is developed to more closely mimic the protein's native environment.•Phenothiazines inhibit both enzymes, though with different potencies.•Phenothiazines are shown to be also uncouplers of oxidative phosphorylation.
The development of macroscopic nanofiber orientation and microscopic crystallite and molecular chain orientation have been investigated during uniaxial stretching of electrospun poly(vinyl alcohol) ...(PVA) non-woven nanofiber mats. Scanning electron microscopy and stress-strain/small-angle X-ray scattering show that the macroscopic nanofiber orientation significantly increases during the initial stage of deformation, and approaches a plateau on the way of stretching. Detailed analyses of the stress-strain/wide-angle X-ray diffraction measurement and polarized Fourier transform infrared spectroscopy indicate that the microscopic crystallite and molecular chain orientation rapidly increase at the initial stage of stretching due to macroscopic nanofiber orientation. At higher deformation, the microscopic modes of orientation continuously develop as a result of the nanofiber stretching. The complicated deformation process of non-woven nanofiber mats is discussed in terms of macroscopic nanofiber orientation and the microscopic crystallite and molecular chain orientation.
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Type II NADH-quinone oxidoreductase (NDH-2) catalyzes the transfer electrons from NADH to the quinone pool and plays an essential role in the oxidative phosphorylation system of Mycobacterium ...tuberculosis (Mtb). The absence of NDH-2 in the mammalian mitochondrial electron transport chain makes this enzyme an attractive target for antibiotic development. To fully establish the kinetic properties of this enzyme, we studied the interaction of Mtb NDH-2 with substrates, NADH, and various quinone analogues and their products in both membrane and soluble environments. These studies, and comparative analyses of the kinetics with thio-NAD+ and quinone electron acceptors, provided evidence that Mtb NDH-2 catalyzes the transfer electrons from NADH to quinone substrates by a nonclassical, two-site ping-pong kinetic mechanism whereby substrate quinones bind to a site that is distinct from the NADH-binding site. Furthermore, the effects of quinols on Mtb NDH-2 catalytic activity demonstrate the presence of two binding sites for quinone ligands, one favoring the reduced form and the other favoring the oxidized form.
Mycobacterium tuberculosis (Mtb) is an obligate aerobe that is capable of long-term persistence under conditions of low oxygen tension. Analysis of the Mtb genome predicts the existence of a branched ...aerobic respiratory chain terminating in a cytochrome bd system and a cytochrome aa3system. Both chains can be initiated with type II NADH:menaquinone oxidoreductase. We present a detailed biochemical characterization of the aerobic respiratory chains from Mtb and show that phenothiazine analogs specifically inhibit NADH:menaquinone oxidoreductase activity. The emergence of drug-resistant strains of Mtb has prompted a search for antimycobacterial agents. Several phenothiazines analogs are highly tuberculocidal in vitro, suppress Mtb growth in a mouse model of acute infection, and represent lead compounds that may give rise to a class of selective antibiotics.
The energy-transducing NADH: quinone (Q) oxidoreductase (complex I) is the largest and most complicated enzyme complex in the oxidative phosphorylation system. Complex I is a redox pump that uses the ...redox energy to translocate H
+ (or Na
+) ions across the membrane, resulting in a significant contribution to energy production. The need to elucidate the molecular mechanisms of complex I has greatly increased. Many devastating neurodegenerative disorders have been associated with complex I deficiency. The structural and functional complexities of complex I have already been established. However, intricate biogenesis and activity regulation functions of complex I have just been identified. Based upon these recent developments, it is apparent that complex I research is entering a new era. The advancement of our knowledge of the molecular mechanism of complex I will not only surface from bioenergetics, but also from many other fields as well, including medicine. This review summarizes the current status of our understanding of complex I and sheds light on new theories and the future direction of complex I studies.
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