Rac1 and Toll-IL-1 receptor domain-containing adapter protein mediate Toll-like receptor 4 induction of HIV-long terminal repeat

Opportunistic infections, common in HIV-1-infected patients, increase HIV replication; however, the intracellular signaling mechanisms involved are not clearly known. We have shown that Toll-like receptor 2 (TLR2), TLR4, and TLR9 mediate microbial Ag-induced HIV-long terminal repeat (HIV-LTR) trans-activation and HIV-1 replication, and that LPS-induced HIV-LTR trans-activation is mediated through myeloid differentiation adapter protein. Recently, Toll-IL-1R domain-containing adapter protein (TIRAP) has been identified as an adapter molecule that mediates responses to TLR2 and TLR4 ligands, and TIRAP was suggested to provide signaling specificity for different TLRs. Rac1, a small GTP-binding protein that is activated upon LPS stimulation of macrophages, activates phosphatidylinositol 3-kinase and Akt and leads to NF-kappaB activation. The roles of Rac1 and TIRAP in LPS activation of HIV replication is not known. In the present study we show that LPS stimulation of human microvessel endothelial cells leads to Rac1 activation. Constitutively active Rac1 (Rac1V12) simulated the effect of LPS to activate HIV-LTR, whereas the expression of dominant negative Rac1 (Rac1N17) partially blocked LPS-induced HIV-LTR trans-activation. Rac1V12-induced HIV-LTR activation was independent of myeloid differentiation adapter protein, and dominant negative TIRAP blocked Rac1V12-induced HIV-LTR trans-activation. In this study we show for the first time that activation of Rac1 leads to HIV-LTR trans-activation, and this is mediated through TIRAP. Together these results underscore the importance of Rac1 and TIRAP in TLR4 activation of HIV replication and help delineate the signaling pathways induced by TLRs to mediate microbial Ag-induced HIV replication and HIV pathogenesis.     

Regulation of cyp26a1 on Th17 cells in mouse peri‐implantation

Cytochrome P450 26A1 (cyp26a1) is expressed in the mouse uterus during peri‐implantation. The repression of this protein is closely associated with a reduction in implantation sites, suggesting a specific role for cyp26a1 in pregnancy and prompting questions concerning how a metabolic enzyme can generate this distinct outcome. To explore the effective downstream targets of cyp26a1 and confirm if its role in peri‐implantation depends on its metabolic substrate RA (retinoic acid), we characterized the changes in the peripheral blood, spleen and uterine implantation sites using the cyp26a1 gene vaccine constructed before. Flow cytometry results showed a significant increase in CD4⁺RORγt⁺ Th17 cells in both the peripheral blood and spleen in the experimental group. The expression of RORγt and IL‐17 presented the Th17 cells reduction in uterus followed by the suppression of cyp26a1 expression. For greater certainty, cyp26a1 antibody blocking model and RNA interference model were constructed to determine the precise target immune cell group. High performance liquid chromatography results showed a significant increase in uterine at‐RA followed by the immunization of cyp26a1 gene vaccine. Both the ascertain by measuring RARα protein levels in peri‐implantation uterus after gene vaccine immunization and researches using the specific agonist and antagonist against RARα suggested that RARα may be the main RA receptor for signal transduction. These results provided more evidence for the signal messenger role of RA in cyp26a1 regulation from the other side. Here, we showed that the cyp26a1‐regulated Th17 cells are dependent on at‐RA signalling, which is delivered through RARα in mouse peri‐implantation.     

Knockdown of phosphotyrosyl phosphatase activator induces apoptosis via mitochondrial pathway and the attenuation by simultaneous tau hyperphosphorylation

Phosphotyrosyl phosphatase activator (PTPA) is decreased in the brains of Alzheimer's disease (AD) and the AD transgenic mouse models. Here, we investigated whether down‐regulation of PTPA affects cell viability and the underlying mechanisms. We found that PTPA was located in the integral membrane of mitochondria, and knockdown of PTPA induced cell apoptosis in HEK293 and N2a cell lines. PTPA knockdown decreased mitochondrial membrane potential and induced Bax translocation into the mitochondria with a simultaneous release of Cyt C, activation of caspase‐3, cleavage of poly (DNA ribose) polymerase (PARP), and decrease in Bcl‐xl and Bcl‐2 protein levels. Over‐expression of Protein phosphatase 2A (PP2A) catalytic subunit (PP2A_C) did not rescue the apoptosis induced by PTPA knockdown, and PTPA knockdown did not affect the level of and their phosphorylation of mitogen‐activated protein kinases (MAPKs), indicating that PP2A and MAPKs were not involved in the apoptosis induced by PTPA knockdown. In the cells with over‐expression of tau, PTPA knockdown induced PP2A inhibition and tau hyperphosphorylation but did not cause significant cell death. These data suggest that PTPA deficit causes apoptotic cell death through mitochondrial pathway and simultaneous tau hyperphosphorylation attenuates the PTPA‐induced cell death.

     

Negative Interference by Rheumatoid Factor of Plasma B-Type Natriuretic Peptide in Chemiluminescent Microparticle Immunoassays

Background

The chemiluminescent microparticle immunoassay (CMIA) is widely used for the quantitative determination of B-type natriuretic peptide (BNP) in human ethylenediaminetetraacetic acid plasma. Rheumatoid factor (RF) is usually thought to result in a positive interference in immunoassays, but it is not clear whether its presence in plasma can lead to interferences in the CMIA of BNP.

Methods

The estimation of BNP recovery was carried out by diluting high-concentration BNP samples with RF-positive or RF-negative plasma at a ratio of 1∶9. The diluted samples were then tested using the ARCHITECT i2000 System and ARCHITECT BNP Reagent Kits and the recovery was then calculated.

Results

When the RF level ranged from 48 to 1420 IU/mL, the average recovery of BNP was 79.29% and 91.61% in the RF-positive and RF-negative plasma samples, respectively, and was thus significantly lower in the group of RF-positive plasma samples than in the group of RF-negative plasma samples. At a dilution of 1∶16, the measured BNP level increased by >36% in six of the seven RF-positive plasma samples. The recovery of BNP increased significantly in the RF-positive plasma samples after pretreatment with IgG-sensitive latex particles. In addition, The BNP recovery was not significantly related to the plasma RF at concentrations ranging from 48 to 2720 IU/mL.

Conclusions

Measurement of BNP by CMIA is susceptible to interference from RF leading to predominantly (but not exclusively) lower results. Pretreatment of samples with blocking reagents is advisable prior to the initiation of denying patient''s necessary treatment.     

Partial Protective Effect of Intranasal Immunization with Recombinant Toxoplasma gondii Rhoptry Protein 17 against Toxoplasmosis in Mice

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that infects a variety of mammals, including humans. An effective vaccine for this parasite is therefore needed. In this study, RH strain T. gondii rhoptry protein 17 was expressed in bacteria as a fusion with glutathione S-transferase (GST) and the recombinant proteins (rTgROP17) were purified via GST-affinity chromatography. BALB/c mice were nasally immunised with rTgROP17, and induction of immune responses and protection against chronic and lethal T. gondii infections were investigated. The results revealed that mice immunised with rTgROP17 produced high levels of specific anti-rTgROP17 IgGs and a mixed IgG1/IgG2a response of IgG2a predominance. The systemic immune response was associated with increased production of Th1 (IFN-γand IL-2) and Th2 (IL-4) cytokines, and enhanced lymphoproliferation (stimulation index, SI) in the mice immunised with rTgROP17. Strong mucosal immune responses with increased secretion of TgROP17-specific secretory IgA (SIgA) in nasal, vaginal and intestinal washes were also observed in these mice. The vaccinated mice displayed apparent protection against chronic RH strain infection as evidenced by their lower liver and brain parasite burdens (59.17% and 49.08%, respectively) than those of the controls. The vaccinated mice also exhibited significant protection against lethal infection of the virulent RH strain (survival increased by 50%) compared to the controls. Our data demonstrate that rTgROP17 can trigger strong systemic and mucosal immune responses against T. gondii and that ROP17 is a promising candidate vaccine for toxoplasmosis.     

A real-time fluorescence polarization activity assay to screen for inhibitors of bacterial ribonuclease P

Ribonuclease P (RNase P) is an essential endonuclease that catalyzes the 5′ end maturation of precursor tRNA (pre-tRNA). Bacterial RNase P is an attractive potential antibacterial target because it is essential for cell survival and has a distinct subunit composition compared to the eukaryal counterparts. To accelerate both structure-function studies and discovery of inhibitors of RNase P, we developed the first real-time RNase P activity assay using fluorescence polarization/anisotropy (FP/FA) with a 5′ end fluorescein-labeled pre-tRNA^Asp substrate. This FP/FA assay also detects binding of small molecules to pre-tRNA. Neomycin B and kanamycin B bind to pre-tRNA^Asp with a K_d value that is comparable to their IC₅₀ value for inhibition of RNase P, suggesting that binding of these antibiotics to the pre-tRNA substrate contributes to the inhibitory activity. This assay was optimized for high-throughput screening (HTS) to identify specific inhibitors of RNase P from a 2880 compound library. A natural product derivative, iriginol hexaacetate, was identified as a new inhibitor of Bacillus subtilis RNase P. The FP/FA methodology and inhibitors reported here will further our understanding of RNase P molecular recognition and facilitate discovery of antibacterial compounds that target RNase P.     

TRPV1 activation impedes foam cell formation by inducing autophagy in oxLDL-treated vascular smooth muscle cells

Vascular smooth muscle cells (VSMCs) are an important origin of foam cells besides macrophages. The mechanisms underlying VSMC foam cell formation are relatively little known. Activation of transient receptor potential vanilloid subfamily 1 (TRPV1) and autophagy have a potential role in regulating foam cell formation. Our study demonstrated that autophagy protected against foam cell formation in oxidized low-density lipoprotein (oxLDL)-treated VSMCs; activation of TRPV1 by capsaicin rescued the autophagy impaired by oxLDL and activated autophagy–lysosome pathway in VSMCs; activation of TRPV1 by capsaicin impeded foam cell formation of VSMCs through autophagy induction; activation of TRPV1 by capsaicin induced autophagy through AMP-activated protein kinase (AMPK) signaling pathway. This study provides evidence that autophagy plays an important role in VSMC foam cell formation and highlights TRPV1 as a promising therapeutic target in atherosclerosis.     

Leaf morphology and cuticular features of Sphenophyllum in the Gigantopteris flora from South China

Permian specimens of Sphenophyllum with preserved cuticular anatomy have been discovered in Cathaysia and have prompted a detailed re-evaluation of the genus in the Gigantopteris flora in South China. New specimens are described and previously published material is re-examined to clarify taxonomic diversity and to establish geographic and stratigraphic ranges for each species. Recognized taxa include Sphenophyllum apiciserratum sp. nov., S. koboense Kobatake, S. meridionale sp. nov., S. minor (Sterzel) Gu and Zhi, S. sinocoreanum Yabe, S. cf. sinocoreanum Yabe, and S. aff. speciosum (Royle) McClelland. Cuticles of Sphenophyllum apiciserratum and S. koboense are hypostomatic with randomly disposed ordinary cells in intercostal regions. Specific concepts are clarified and unresolved taxonomic problems are discussed. The South China taxa are compared with those from Euramerican, Gondwanan and Angaran floras, and the South China Cathaysian species are found to be taxonomically distinct.