Rotating pressure measurement system using an on board calibration standard

A computer-controlled multichannel pressure measurement system was developed to acquire detailed flow field measurements on board the Large Low Speed Centrifugal Compressor Research Facility at the NASA Lewis Research Center. A pneumatic slip ring seal assembly is used to transfer calibration pressures to a reference standard transducer on board the compressor rotor in order to measure very low differential pressures with the high accuracy required. A unique data acquisition system was designed and built to convert the analog signal from the reference transducer to the variable frequency required by the multichannel pressure measurement system and also to provide an output for temperature control of the reference transducer. The system also monitors changes in test cell barometric pressure and rotating seal leakage and provides an on screen warning to the operator if limits are exceeded. The methods used for the selection and testing of the the reference transducer are discussed, and the data acquisition system hardware and software design are described. The calculated and experimental data for the system measurement accuracy are also presented

Nuclear Factor-κB-Independent Anti-Inflammatory Action of Salicylate in Human Endothelial Cells

In contrast to aspirin, salicylate, its active metabolite, possesses profound anti-inflammatory properties without blocking cyclooxygenase. Inhibition of the transcription factor nuclear factor-κB (NF-κB) has been discussed to play a role in the anti-inflammatory profile of salicylate. However, NF-κB-independent effects of salicylate have been assumed but have up to now been poorly investigated. Therefore, the aim of the present study was to investigate NF-κB-independent anti-inflammatory mechanisms of salicylate in human umbilical vein endothelial cells using interleukin-4 (IL-4) as NF-κB-independent proinflammatory stimulus and P-selectin as inflammatory read-out parameter. Using quantitative real-time reverse transcriptionpolymerase chain reaction, we found that salicylate decreases IL-4-induced P-selectin expression. As judged by Western blot analysis, salicylate increased endothelial heme oxygenase-1 (HO-1) protein levels. Using both the HO-1 inhibitor tin(II) protoporphyrin IX and HO-1 antisense oligonucleotides, we causally linked the induction of HO-1 to the decrease of P-selectin. Moreover, we were interested in the signaling mechanisms leading to the up-regulation of HO-1 by salicylate. c-Jun NH2-terminal kinase (JNK) was found to be activated by salicylate, and we could causally link this activation to the induction of HO-1 by using the JNK inhibitor 1,9-pyrazoloanthrone. By applying activator protein-1 (AP-1) decoys, it was shown that the transcription factor AP-1 is crucially involved in the up-regulation of HO-1 downstream of JNK. In summary, our study introduces HO-1 as novel NF-κB-independent anti-inflammatory target of salicylate in human endothelial cells. Moreover, we elucidated the JNK/AP-1 pathway as crucial for the induction of HO-1 by salicylate

Translational independence between overlapping genes for a restriction endonuclease and its transcriptional regulator

<p>Abstract</p> <p>Background</p> <p>Most type II restriction-modification (RM) systems have two independent enzymes that act on the same DNA sequence: a modification methyltransferase that protects target sites, and a restriction endonuclease that cleaves unmethylated target sites. When RM genes enter a new cell, methylation must occur before restriction activity appears, or the host's chromosome is digested. Transcriptional mechanisms that delay endonuclease expression have been identified in some RM systems. A substantial subset of those systems is controlled by a family of small transcription activators called C proteins. In the PvuII system, C.PvuII activates transcription of its own gene, along with that of the downstream endonuclease gene. This regulation results in very low R.PvuII mRNA levels early after gene entry, followed by rapid increase due to positive feedback. However, given the lethal consequences of premature REase accumulation, transcriptional control alone might be insufficient. In C-controlled RM systems, there is a ± 20 nt overlap between the C termination codon and the R (endonuclease) initiation codon, suggesting possible translational coupling, and in many cases predicted RNA hairpins could occlude the ribosome binding site for the endonuclease gene.</p> <p>Results</p> <p>Expression levels of <it>lacZ </it>translational fusions to <it>pvuIIR </it>or <it>pvuIIC </it>were determined, with the native <it>pvuII </it>promoter having been replaced by one not controlled by C.PvuII. In-frame <it>pvuIIC </it>insertions did not substantially decrease either <it>pvuIIC-lacZ </it>or <it>pvuIIR-lacZ </it>expression (with or without C.PvuII provided <it>in trans</it>). In contrast, a frameshift mutation in <it>pvuIIC </it>decreased expression markedly in both fusions, but mRNA measurements indicated that this decrease could be explained by transcriptional polarity. Expression of <it>pvuIIR-lacZ </it>was unaffected when the <it>pvuIIC </it>stop codon was moved 21 nt downstream from its WT location, or 25 or 40 bp upstream of the <it>pvuIIR </it>initiation codon. Disrupting the putative hairpins had no significant effects.</p> <p>Conclusions</p> <p>The initiation of translation of <it>pvuIIR </it>appears to be independent of that for <it>pvuIIC</it>. Direct tests failed to detect regulatory rules for either gene overlap or the putative hairpins. Thus, at least during balanced growth, transcriptional control appears to be sufficiently robust for proper regulation of this RM system.</p

Varying effects of temperature, Ca2+ and cytochalasin on fusion activity mediated by human immunodeficiency virus type 1 and type 2 glycoproteins

AbstractWe examined fusion mediated by the human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) envelope glycoproteins under various experimental conditions. Incubation of HeLa cells expressing HIV-2ROD and HIV-2SBL/ISY envelope glycoproteins with HeLa-CD4 target cells resulted in fusion at temperatures ≥25°C whereas fusion with cells expressing HIV-1Lai occurred only at ≥31°C. HIV-2 envelope glycoprotein-mediated fusion proceeded in the absence of Ca2+ in the culture medium, whereas HIV-1 fusion required Ca2+ ions for fusion. In contrast to HIV-2 envelope glycoprotein fusion, incubations in the presence of the 0.5 μM cytochalasin B completely inhibited HIV-1 envelope glycoprotein-mediated fusion. Our results suggest that in contrast to HIV-2, HIV-1 fusion is dependent on dynamic processes in the target membrane

Photo-activation of the hydrophobic probe iodonaphthylazide in cells alters membrane protein function leading to cell death

<p>Abstract</p> <p>Background</p> <p>Photo-activation of the hydrophobic membrane probe 1, 5 iodonaphthylazide (INA) by irradiation with UV light (310–380 nm) results in the covalent modification of transmembrane anchors of membrane proteins. This unique selectivity of INA towards the transmembrane anchor has been exploited to specifically label proteins inserted in membranes. Previously, we have demonstrated that photo-activation of INA in enveloped viruses resulted in the inhibition of viral membrane protein-induced membrane fusion and viral entry into cells. In this study we show that photo-activation of INA in various cell lines, including those over-expressing the multi-drug resistance transporters MRP1 or Pgp, leads to cell death. We analyzed mechanisms of cell killing by INA-UV treatment. The effects of INA-UV treatment on signaling via various cell surface receptors, on the activity of the multi-drug resistance transporter MRP1 and on membrane protein lateral mobility were also investigated.</p> <p>Results</p> <p>INA treatment of various cell lines followed by irradiation with UV light (310–380 nm) resulted in loss of cell viability in a dose dependent manner. The mechanism of cell death appeared to be apoptosis as indicated by phosphatidylserine exposure, mitochondrial depolarization and DNA fragmentation. Inhibition by pan-caspase inhibitors and cleavage of caspase specific substrates indicated that at low concentrations of INA apoptosis was caspase dependent. The INA-UV treatment showed similar cell killing efficacy in cells over-expressing MRP1 function as control cells. Efflux of an MRP1 substrate was blocked by INA-UV treatment of the MRP1-overexpressing cells. Although INA-UV treatment resulted in inhibition of calcium mobilization triggered by chemokine receptor signaling, Akt phosphorylation triggered by IGF1 receptor signaling was enhanced. Furthermore, fluorescence recovery after photobleaching experiments indicated that INA-UV treatment resulted in reduced lateral mobility of a seven transmembrane G protein-coupled receptor.</p> <p>Conclusion</p> <p>INA is a photo-activable agent that induces apoptosis in various cancer cell lines. It reacts with membrane proteins to alter the normal physiological function resulting in apoptosis. This activity of INA maybe exploited for use as an anti-cancer agent.</p