Evaluating real-world CO2 and NOX emissions for public transit buses using a remote wireless on-board diagnostic (OBD) approach.

The challenge to mitigate real-world emissions from vehicles calls for powerful in-use compliance supervision. The remote on-board diagnostic (OBD) approach, with wireless data communications, is one of the promising next-generation monitoring methods. We collected second-by-second profiles of carbon dioxide (CO2) and nitrogen oxides (NOX) emissions, driving conditions and engine performance for three conventional diesel and three hybrid diesel buses participating in a remote OBD pilot program in Nanjing, China. Our results showed that the average CO2 emissions for conventional diesel and hybrid diesel buses were 816 ± 83 g km-1 and 627 ± 54 g km-1, respectively, under a typical driving pattern. An operating mode binning analysis indicated that CO2 emissions reduction by series-parallel hybrid technology was largely because of the significant benefits of the technology under the modes of low speed and low power demand. However, significantly higher CO2 emissions were observed for conventional diesel buses during rush hours, higher than 1200 g km-1. The OBD data suggested no improvement in NOX emission reduction for hybrid buses compared with conventional buses; both were approximately 12 g km-1 because of poor performance of the selective catalyst reduction (SCR) systems in the real world. Speed-dependent functions for real-world CO2 and NOX emissions were also constructed. The CO2 emissions of hybrid buses were much less sensitive to the average speed than conventional buses. If the average speed decreased from 20 km h-1 to 10 km h-1, the estimated CO2 emission factor for conventional buses would be increased by 34%. Such a change in speed would increase NOX emissions for conventional and hybrid buses by 38% and 56%, respectively. This paper demonstrates the useful features of the remote OBD system and can inform policy makers how to take advantage of these features in monitoring in-use vehicles.

Leader sequence is required for activity of transmembrane tumor necrosis factor-alpha.

Transmembrane tumor necrosis factor alpha (tmTNF-alpha) has a variety of biological activities different from soluble TNF-alpha (sTNF-alpha), but the only difference in sequence is its leader sequence (LS). To investigate the effect of the LS on tmTNF-alpha activity, single amino acid substitutions in the LS and its linked extracellular mature domain were made in an in vitro translation system and in an intact cell system. Mutations at Met(-71) and Cys(-28) in the LS obliterated cytotoxicity of tmTNF-alpha, whilst their secretory form retained full activity compared to parental sTNF-alpha. The lost cytotoxicity of Met(-71) mutant tmTNF-alpha was partly due to a reduced receptor binding activity. In spite of full receptor binding activity, Cys(-28) mutant tmTNF-alpha failed to induce NO production and iNOS mRNA transcription via forward signaling, but synergized with sTNF-alpha in IL-8 mRNA transcription via reverse signaling. The Asp(143) mutant tmTNF-alpha lost the ability to bind TNFR and to kill MCF-7 cells, whilst its secretory form retained about 60% cytotoxicity of parental sTNF-alpha. Although the mutation at Phe(87) had full activity in both forms, its membrane form induced a change in cell death mode from apoptosis to necrosis, in contrast to wild-type TNF-alpha whose membrane molecule chiefly induced apoptosis and secretory molecule mainly caused necrosis in MCF-7, respectively. The data suggest that the LS may be required for maintaining the correct structure and the bioactivity of tmTNF-alpha.

Influence of reverse signaling via membrane TNF-alpha on cytotoxicity of NK92 cells.

Membrane tumor necrosis factor-alpha (mTNF-alpha) serves as a receptor transducing signals into mTNF-alpha-bearing cells. Among human peripheral blood mononuclear cells, natural killer (NK) cells have been reported to be the only cell type constitutively expressing mTNF-alpha, which is involved in the cytotoxicity of resting NK cells. Using an IL-2-dependent human NK cell line, NK92, which constitutively expresses mTNF-alpha, we examined the effect of reverse signaling via mTNF-alpha on cellular activities. When the cells were prestimulated with soluble TNFR1 (sTNFR1) which activated mTNF-alpha-mediated reverse signaling, the cytotoxicity of NK92 cells was significantly increased. Further investigation demonstrated that prestimulation with sTNFR1 augmented exocytosis and mRNA transcription of two cytotoxic molecules, perforin and granzyme B, which could serve as underlying molecular mechanisms by which mTNF-alpha-mediated reverse signaling promoted cytotoxicity of NK cells toward K562 cells. On the other hand, pretreatment of NK92 with sTNFR1 boosted the expression of FasL and TNF-alpha, including both the secretory and membrane forms. These molecules also contribute to the NK-mediated cytotoxicity, although K562 cells are Fas-negative and sTNF-alpha-resistant. Interestingly, the mTNF-alpha reverse signaling was found to act synergistically with IL-2 on NK-mediated cytotoxicity. This synergy markedly promoted the production of secretory as well as membrane cytotoxic molecules which may be responsible for the enhanced NK92-mediated cytotoxicity. Our observations suggest that, via reverse signaling, constitutively expressed mTNF-alpha may sensitize NK cells to activating stimuli, such as IL-2, resulting in increased NK-mediated cytotoxicity through promoting the production of multiple cytotoxic effector molecules.

The anti-tumor effect of human monocyte-derived dendritic cells loaded with HSV-TK/GCV induced dying cells.

Herpes simplex virus thymidine kinase (HSV-TK) gene and dendritic cells (DC) have been used as the pioneering in cancer therapy. HSV-TK gene can induce apoptosis and necrosis in tumor cells in the presence of the non-toxic prodrug ganciclovir (GCV). We investigated the anti-tumor effect of DC vaccination by introducing dying cells from HSV-TK gene treatment as an adjuvant. HepG(2)-TK cell line was established by transfecting human hepatoma cell line HepG(2) (HLA-A(2) positive) with HSV-TK gene. Dying tumor cells were generated by culturing HepG(2)-TK cells with GCV. After engulfed dying cells efficiently, immature DCs (imDC) derived from human monocytes were fully matured and elicited marked proliferation and cytotoxicity against HLA matched HepG(2) cells in autologous peripheral blood mononuclear cells (PBMC). It also implied that HepG(2) specific CTLs played an important role in the cytotoxicity which was primarily depended on Th1 responses. Given the feasibility of inducing dying cells by HSV-TK/GCV in vivo, our results suggest an effective method in clinical human hepatocellular carcinoma (HCC) treatment by an in vitro model of applying HSV-TK gene modified human tumor cells integrated with DC vaccination.

Mechanism of homocysteine-induced Rac1/NADPH oxidase activation in mesangial cells: role of guanine nucleotide exchange factor Vav2.

We have demonstrated that homocysteine (Hcys) stimulates de novo ceramide synthesis and thereby induces NADPH oxidase activation by increase of Rac GTPase activity in rat mesangial cells (RMCs). However, which isofrom of Rac GTPases is involved in Hcys-induced NADPH oxidase activity and what mechanism mediates Hcys-induced Rac GTPase activation remain unknown. The present study first addressed the role of Rac1 and then determined the contribution of a subfamily of Guanine Nucleotide Exchange Factors (GEFs), Vav, to the action of Hcys on Rac and NADPH oxidase activities in RMCs. By small interfering RNA (siRNA), it was found that Rac1-siRNA attenuated Hcys-induced superoxide (O(2)(-)) production. To explore the mechanism activating Rac by Hcys, GEF-Vav was examined. Vav2 was found to be a predominant isoform among Vav family in RMCs. In Vav2-siRNA transfected RMCs, Hcys-induced Rac activity was blocked, which was accompanied by significant reduction of Hcys-induced O(2)(-). production. This Vav2-siRNA also blocked Rac activation induced by C16-Ceramide (C16-Cer), an intermediate lipid product stimulated by Hcys. Furthermore, we found that Hcys induced Vav2 phosphorylation in a time-dependent manner, which could be induced by C16-Cer and blocked by inhibition of de novo ceramide synthesis. These results suggest that Vav2 importantly contributes to Hcys-induced increase in Rac1 activity and consequent activation of NADPH oxidase in RMCs via ceramide-associated tyrosine phosphorylation.

Acid sphingomyelinase and its redox amplification in formation of lipid raft redox signaling platforms in endothelial cells.

This study examined the role of acid sphingomyelinase (ASM) and its redox amplification in mediating the formation of lipid raft (LR) redox signaling platforms in coronary arterial endothelial cells (CAECs). Using small interference RNA (siRNA) of ASM, Fas ligand (FasL)-induced increase in ASM activity, production of ceramide, and LR clustering in CAECs were blocked, and clustered Fas was also substantially reduced in detergent-resistant membrane fractions of CAECs. LR clustering, gp91(phox) aggregation, and p47(phox) translocation to the LR clusters induced by FasL were also blocked in ASM-siRNA transfected CAECs. Corresponding to this reduction of LR clustering with NAD(P)H oxidase subunits in ASM-siRNA transfected CAECs, superoxide (O(2)(-*)) production was significantly decreased as measured by either ESR or fluorescent spectrometry. Interestingly, superoxide dismutase (SOD) not only scavenged (O(2)(-*)), but also markedly attenuated LR clustering. Xanthine/xanthine oxidase, an exogenous (O(2)(-*)) generating system, dramatically increased ASM activity and LR clustering in EC membrane and enhanced FasL-induced LR clustering, which were blocked by SOD. These results suggest that that ASM activates LR clustering to form redox signaling platforms, where (O(2)(-*)) production enhances ASM activity, and thereby results in a forwarding amplification of LR and redox signaling. This ASM-mediated feedforwarding mechanism may be critical for an efficient transmembrane signaling through LRs.

Podocyte injury and glomerulosclerosis in hyperhomocysteinemic rats.

BACKGROUND/AIMS: We previously reported that increase in plasma homocysteine (Hcys) levels by a 6-week methionine treatment produced remarkable glomerular injury. However, the mechanism by which hyperhomocysteinemia (hHcys) produces glomerular injury remains unknown. The present study was to observe when glomerular injury happens during hHcys and to explore the possible role of podocyte injury in the progression of glomerulosclerosis associated with hHcys. METHODS: Uninephrectomized Sprague-Dawley rats treated with methionine were used to examine the time course of glomerular injury induced by hHcys. RESULTS: Creatinine clearance was not different until rats were treated with methionine for 6 weeks, although plasma Hcys levels significantly increased at the 1st week of methionine treatment. However, urinary albumin excretion increased at the 2nd week of methionine treatment. Morphological examinations showed that mesangial expansion occurred at the 2nd week and podocyte effacement was also observed as processed glomerular damage during hHcys. Immunofluorescence analyses demonstrated that podocin and nephrin expressions were reduced, while alpha-actinin-4 increased during hHcys. CONCLUSIONS: Increased plasma Hcys level is an important pathogenic factor resulting in glomerular injury even in the very early time of hHcys. These pathogenic effects of Hcys are associated with podocyte injury and changed expression and distribution of podocyte-associated proteins.

[Detection of interaction of binding affinity of aromatic hydrocarbon receptor to the specific DNA by exonuclease protection polymerase chain reaction assay].

OBJECTIVE: To establish an exonuclease protection mediated polymerase chain reaction (PCR) assay for the non-radioactive, sensitive detection of the binding of protein and DNA. METHODS: The 1 pmol/L-10 nmol/L 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dissolved in dimethyl sulphoxide (DMSO), was added into 100 microl SD rat hepatic cytosol in vitro, which contained different amount of aromatic hydrocarbon receptors (AhR) and relative proteins, and ligand-AhR-DRE complex were formed in addition to 1 fmol/L-100 nmol/L DNAs probes containing the sequence of DRE. With the digestion of Exonuclease III and S1 nuclease, free DNAs were digested to oligonucleotide and binding DNA remained due to protein (AhR) protection and be amplified by PCR. The results of PCRs were shown by loading on 2% agarose electrophoresis. DMSO was used as negative control and blank control was set up. RESULTS: Target DNA (285 bp) could be observed in the ligand groups, but not in the control group. The minimal amount of receptor was 2.5 fmol/L and the minimal amount of DNA probes was 2 fmol. CONCLUSIONS: Exonuclease protection mediated PCR assay should be a good non-radioactive tool to quantify the interaction of protein and DNA with high sensitivity and simplicity.