Intracellular trafficking of HIV-1 Gag via Syntaxin 6-positive compartments/vesicles: Involvement in tumor necrosis factor secretion.

HIV-1 Gag protein is synthesized in the cytosol and is transported to the plasma membrane, where viral particle assembly and budding occur. Endosomes are alternative sites of Gag accumulation. However, the intracellular transport pathways and carriers for Gag have not been clarified. We show here that Syntaxin6 (Syx6), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane fusion in post-Golgi networks, is a molecule responsible for Gag trafficking and also for tumor necrosis factor-α (TNFα) secretion and that Gag and TNFα are cotransported via Syx6-positive compartments/vesicles. Confocal and live-cell imaging revealed that Gag colocalized and cotrafficked with Syx6, a fraction of which localizes in early and recycling endosomes. Syx6 knockdown reduced HIV-1 particle production, with Gag distributed diffusely throughout the cytoplasm. Coimmunoprecipitation and pulldown show that Gag binds to Syx6, but not its SNARE partners or their assembly complexes, suggesting that Gag preferentially binds free Syx6. The Gag matrix domain and the Syx6 SNARE domain are responsible for the interaction and cotrafficking. In immune cells, Syx6 knockdown/knockout similarly impaired HIV-1 production. Interestingly, HIV-1 infection facilitated TNFα secretion, and this enhancement did not occur in Syx6-depleted cells. Confocal and live-cell imaging revealed that TNFα and Gag partially colocalized and were cotransported via Syx6-positive compartments/vesicles. Biochemical analyses indicate that TNFα directly binds the C-terminal domain of Syx6. Altogether, our data provide evidence that both Gag and TNFα make use of Syx6-mediated trafficking machinery and suggest that Gag expression does not inhibit but rather facilitates TNFα secretion in HIV-1 infection.

Fusion of parvovirus B19 receptor-binding domain and pneumococcal surface protein A induces protective immunity against parvovirus B19 and Streptococcus pneumoniae.

BACKGROUND: Parvovirus B19 (B19) is a well-known cause of fifth disease in children, but infection during pregnancy may cause hydrops fetalis and stillbirth. The receptor-binding domain (RBD) of the VP1 unique capsid plays a pivotal role in infection. Here, we aimed to improve the immunogenicity of an RBD-based vaccine by genetically fusing it with Streptococcus pneumoniae surface protein A (PspA). METHODS: Mice were intramuscularly injected with RBD-based vaccines. Antigen-specific antibodies and neutralizing activity against B19 were measured. Protective immunity against S. pneumoniae was evaluated by monitoring the survival of mice nasally challenged with bacteria and determining antigen-specific T cell activation in splenic cells. RESULTS: RBD alone failed to generate neutralizing antibodies against B19, but fusion with PspA induced higher levels of neutralizing IgG compared to B19 virus-like particles. Furthermore, a comparable level of PspA-specific IgG was induced by RBD-PspA and PspA alone, which was sufficient to protect mice against pneumococcal infection. Stimulation with PspA, but not RBD, induced cytokine production in splenic cells from mice immunized with RBD-PspA, suggesting that PspA-specific T cells supported immunoglobulin class switching of both RBD- and PspA-specific B cells. CONCLUSIONS: RBD-PspA should be an effective bivalent vaccine against B19 and S. pneumoniae infections.

An Experimental Investigation on Micro End Milling with High-Speed Up Cut Milling for Hardened Die Steel.

The importance of micromachining using small diameter end mills and the dies used for them has been increasing in the machining of small parts. However, the reality is that there are various requirements to improve the machining surface, machining accuracy, machining efficiency, and tool life. Therefore, this paper discusses the possibility of satisfying these requirements by high-speed up cut milling in side cutting. The goal of this study was to solve the aforementioned problems, by conducting a detailed analysis of the machining phenomena in order to understand their mechanisms. In particular, the effects of high-speed cutting using a high-speed air-turbine spindle with highly stiff rolling bearings were analyzed. Moreover, cutting experiments were conducted by measuring the cutting force and flank wear of the tool, to reveal the differences in the cutting phenomena relative to the cutting direction in high-speed micro end milling. Description of the machined surface and the measurement of its profile were also included in the discussions. On the basis of the results, high-speed up cut milling is a better choice than down cut milling; furthermore, a high-feed rate further increases machining efficiency and improves tool life.

High-contrast photoswitching of nonlinear optical response in crosslinked ferroelectric liquid-crystalline polymers.

A conceptually novel materials design, based on crosslinked ferroelectric liquid-crystalline polymers, is demonstrated for efficient switching of a second-order nonlinear optical (NLO) response in the solid state. By controlling the molecular alignment of the NLO moieties through two-photon isomerization of azobenzene molecules, reversible isothermal photocontrol of second-harmonic generation is achieved with contrast of up to 20.

Glutamic acid residues of bacteriorhodopsin at the extracellular surface as determinants for conformation and dynamics as revealed by site-directed solid-state 13C NMR.

We recorded (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled bacteriorhodopsin (bR) and a variety of its mutants, E9Q, E74Q, E194Q/E204Q (2Glu), E9Q/E194Q/E204Q (3Glu), and E9Q/E74Q/E194Q/E204Q (4Glu), to clarify contributions of the extracellular (EC) Glu residues to the conformation and dynamics of bR. Replacement of Glu-9 or Glu-74 and Glu-194/204 at the EC surface by glutamine(s) induced significant conformational changes in the cytoplasmic (CP) surface structure. These changes occurred in the C-terminal alpha-helix and loops, and also those of the EC surface, as viewed from (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled proteins. Additional conformational changes in the transmembrane alpha-helices were induced as modified retinal-protein interactions for multiple mutants involving the E194Q/E204Q pair. Significant dynamic changes were induced for the triple or quadruple mutants, as shown by broadened (13)C NMR peaks of [1-(13)C]Val-labeled proteins. These changes were due to acquired global fluctuation motions of the order of 10(-4)-10(-5) s as a result of disorganized trimeric form. In such mutants (13)C NMR signals from Val residues of [1-(13)C]Val-labeled triple and quadruple mutants near the CP and EC surfaces (including 8.7-A depth from the surface) were substantially suppressed, as shown by comparative (13)C NMR studies with and without 40 micro M Mn(2+) ion. We conclude that these Glu residues at the EC surface play an important role in maintaining the native secondary structure of bR in the purple membrane.