Coregulation of NADPH oxidase activation and phosphorylation of a 48-kD protein(s) by a cytosolic factor defective in autosomal recessive chronic granulomatous disease.

The mechanisms regulating activation of the respiratory burst enzyme, NADPH oxidase, of human neutrophils (PMN) are not yet understood, but protein phosphorylation may play a role. We have utilized a defect in a cytosolic factor required for NADPH oxidase activation observed in two patients with the autosomal recessive form of chronic granulomatous disease (CGD) to examine the role of protein phosphorylation in activation of NADPH oxidase in a cell-free system. NADPH oxidase could be activated by SDS in reconstitution mixtures of cytosolic and membrane subcellular fractions from normal PMN, and SDS also enhanced phosphorylation of at least 16 cytosolic and 14 membrane-associated proteins. However, subcellular fractions from CGD PMN plus SDS expressed little NADPH oxidase activity, and phosphorylation of a 48-kD protein(s) was selectively defective. The membrane fraction from CGD cells could be activated for NADPH oxidase when mixed with normal cytosol and phosphorylation of the 48-kD protein(s) was restored. In contrast, the membrane fraction from normal cells expressed almost no NADPH oxidase activity when mixed with CGD cytosol, and phosphorylation of the 48-kD protein(s) was again markedly decreased. Protein kinase C (PKC) activity in PMN from the two patients appeared to be normal, suggesting that a deficiency of PKC is not the cause of the defective 48-kD protein phosphorylation and that the cytosolic factor is not PKC. These results demonstrate that the cytosolic factor required for activation of NADPH oxidase also regulates phosphorylation of a specific protein, or family of proteins, at 48 kD. Although the nature of this protein(s) is still unknown, it may be related to the functional and phosphorylation defects present in CGD PMN and to the activation of NADPH oxidase in the cell-free system.

Mechanisms of free radical oxidation of unsaturated lipids.

The primary products formed from the autoxidation of lipids can be understood based upon a mechanism that involves five different reaction types. These reactions are: reaction of a carbon radical and molecular oxygen, atom transfer of a hydrogen from substrate to the chain carrying peroxyl, fragmentation of the chain carrying peroxyl to give oxygen and a carbon radical, rearrangement of the peroxyl, and cyclization of the peroxyl. The mechanism of these primary reaction steps has been the focus of extensive research over the past fifty years, and the current level of understanding of these transformations is the subject of this review.

Monte Carlo validation experiments for the gas Cherenkov detectors at the National Ignition Facility and Omega.

The gas Cherenkov detectors at NIF and Omega measure several ICF burn characteristics by detecting multi-MeV nuclear γ emissions from the implosion. Of primary interest are γ bang-time (GBT) and burn width defined as the time between initial laser-plasma interaction and peak in the fusion reaction history and the FWHM of the reaction history respectively. To accurately calculate such parameters the collaboration relies on Monte Carlo codes, such as GEANT4 and ACCEPT, for diagnostic properties that cannot be measured directly. This paper describes a series of experiments performed at the High Intensity γ Source (HIγS) facility at Duke University to validate the geometries and material data used in the Monte Carlo simulations. Results published here show that model-driven parameters such as intensity and temporal response can be used with less than 50% uncertainty for all diagnostics and facilities.

Mach-Zehnder recording systems for pulsed power diagnostics.

Fiber-optic transmission and recording systems, based on Mach-Zehnder modulators, have been developed and installed at the National Ignition Facility (NIF), and are being developed for other pulsed-power facilities such as the Z accelerator at Sandia, with different requirements. We present the design and performance characteristics for the mature analog links, based on the system developed for the Gamma Reaction History diagnostic at the OMEGA laser and at NIF. For a single detector channel, two Mach-Zehnders are used to provide high dynamic range at the full recording bandwidth with no gaps in the coverage. We present laboratory and shot data to estimate upper limits on the radiation effects as they impact recorded data quality. Finally, we will assess the technology readiness level for mature and developing implementations of Mach-Zehnder links for these environments.

Development and characterization of sub-100 ps photomultiplier tubes.

We describe the evaluation of a microchannel plate (MCP) photomultiplier tube (PMT), incorporating a 3 µm pore MCP and constant voltage anode and cathode gaps. The use of the small pore size results in PMTs with response functions of the order of 85 ps full-width-half-maximum, while the constant electric field across the anode and cathode gaps produces a uniform response function over the entire operating range of the device. The PMT was characterized on a number of facilities and employed on gas Cherenkov detectors fielded on various deuterium tritium fuel (DT) implosions on the Omega Laser Facility at the University of Rochester. The Cherenkov detectors are part of diagnostic development to measure Gamma ray reaction history for DT implosions on the National Ignition Facility.

GEANT4 simulations of Cherenkov reaction history diagnostics.

This paper compares the results from a GEANT4 simulation of the gas Cherenkov detector 1 (GCD1) with previous simulations and experimental data from the Omega laser facility. The GCD1 collects gammas emitted during a deuterium-tritium capsule implosion and converts them, through several processes, to Cherenkov light. Photon signals are recorded using subnanosecond photomultiplier tubes, producing burn reaction histories. The GEANT4 GCD1 simulation is first benchmarked against ACCEPT, an integrated tiger series code, with good agreement. The simulation is subsequently compared with data from the Omega laser facility, where experiments have been performed to measure the effects of Hohlraum materials on reaction history signals, in preparation for experiments at the National Ignition Facility.

Interaction of Sendai virus proteins with the cytoplasmic surface of erythrocyte membranes following viral envelope fusion.

Radiolabeled Sendai virus was allowed to fuse with human erythrocytes and inside out vesicles were prepared from the erythrocyte membranes. Viral proteins incorporated into inside out vesicles were released from the membrane following treatment with various agents which perturb protein structure. Disruption products were analyzed by sucrose gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to identify the viral proteins. The results indicated that 6 M guanidine, 40 mM lithium diiodosalicylate (pH 11-13), and 4 M potassium thiocyanate removed viral nucleocapsid and M proteins from the vesicles. Urea (6.0 M) and 5 mM p-chloromercuribenzenesulfonate removed only viral nucleocapsid proteins. KCl (1.0 or 0.1 M), 5 or 0.5 mM sodium phosphate, 10 mM EDTA (pH 10), 10 mM lithium diiodosalicylate, 1 M urea, 0.5 mM ATP, or freeze-thaw cycles did not release any viral proteins. By contrast, treatment of inside out vesicles with Triton X-100 solubilizes the lipid bilayer, releasing viral integral membrane glycoproteins and leaving viral nucleocapsids intact. Nucleocapsids isolated from virions do not adsorb nonspecifically to control inside out vesicles. These data imply that Sendai viral nucleocapsid and M proteins interact extensively with the cytoplasmic surface of infected cell membranes, in the manner of peripheral membrane proteins, whereas the glycoproteins, HN and F1, behave as integral membrane proteins.

Dissociation of newly synthesized Sendai viral proteins from the cytoplasmic surface of isolated plasma membranes of infected cells.

The interaction of Sendai viral proteins with the membranes of infected cells during budding of progeny virions was studied. BHK cells infected with Sendai virus were labeled with [35S]methionine, and the plasma membranes were purified on polycationic polyacrylamide beads. The isolated membranes were incubated with various agents which perturb protein structure to dissociate viral proteins from the membranes. Incubation of membranes with thiocyanate and guanidine removed both the M and nucleocapsid proteins. Urea (6 M) removed the nucleocapsid proteins but removed M protein only in the presence of 0.1 or 1.0 M KCl. In contrast, high salt concentrations alone eluted only the M protein, leaving the nucleocapsid proteins completely membrane bound. About 65% of the M protein was eluted in the presence of 4 M KCl. The remaining membrane-associated M protein was resistant to further extraction by 4 M KCl. Thus, M protein forms two types of interaction with the membrane, one of them being a more extensive association with the membrane than the other.

Alterations in cell protein phosphorylation in human neutrophils exposed to influenza A virus. A possible mechanism for depressed cellular end-stage functions.

When polymorphonuclear leukocytes (PMN) are exposed to most harvests of influenza A virus (depressing virus, DV) for 20 min, chemotactic, secretory, and oxidative functions are depressed upon subsequent exposure to soluble or particulate stimuli. Other harvests of influenza A virus (non-DV) do not alter these activities. The DV-induced changes in multiple functions suggest the virus may interfere with steps involved in PMN activation. Because some of these steps may be regulated by protein phosphorylation, we examined the effect of non-DV and DV on cellular protein phosphorylation. PMN loaded with 32P-labeled inorganic orthophosphate were exposed to non-DV, DV, or buffer for 30 min; cells were then treated with buffer, FMLP (10(-6) M), or PMA (100 ng/ml) for 30 s. Samples were sonicated and centrifuged; cytosolic and particulate fractions were analyzed by SDS-PAGE and autoradiography. Exposure of PMN to either non-DV or DV caused phosphorylation of several cell proteins. However, when DV-treated PMN were then stimulated with FMLP or PMA, further phosphorylation was inhibited compared to non-DV- or buffer-treated cells. This suggests that DV-induced depression of PMN end-stage functions may be due to changes in cell protein phosphorylation. DV could interfere with phosphorylation of PMN proteins by altering protein kinase activity. We therefore examined the influence of non-DV and DV on some parameters that could affect kinase function. PMN intracellular [Ca2+] was monitored by using the fluorescent Ca2+ indicator, Indo 1, and cAMP levels were measured by RIA. PMN treated with DV alone or DV plus FMLP had higher intracellular [CA2+] than PMN similarly treated with non-DV or buffer. Exposure of PMN to non-DV, DV, or buffer caused minimal changes in cAMP levels, and similar increases occurred in cAMP levels upon FMLP stimulation. To determine whether DV interferes with transmembrane signaling, the effect of influenza virus on PMN transmembrane potential was studied by using a fluorescent cyanine dye. Transmembrane potential changes were greater in PMN exposed to DV than to non-DV or buffer; however, subsequent stimulation with FMLP caused equivalent changes in transmembrane potential. Our data show that protein phosphorylation in PMN is induced by DV and non-DV infection; upon subsequent stimulation with FMLP or PMA, there is inhibited cellular phosphorylation only in PMN previously exposed to DV.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of the guanidinobenzoatase in the epididymal fluids of the mouse.

Guanidinobenzoatase (GB), a proteolytic enzyme found in the epididymal fluids of mice, was purified to apparent homogeneity by molecular sieving and affinity chromatography. It has a molecular mass of 71 kDa and its enzymatic activity is heat labile and sensitive to EGTA. Its kinetic parameters (K(m) of 6.66 microM and a Vmax of 4.38 nmol/min/mg) were determined using 4-methylumbelliferyl-p-guanidinobenzoate (MUGB) as the substrate. GB activity is concentrated in the cauda epididymal region of the genital tract. Heat-solubilized whole zonae, biologically active ZP3, and several serine proteinase inhibitors, including a proteinase inhibitor endogenous to the male genital tract, effectively block the ability of GB to hydrolyze MUGB. Pretreating cumulus-free, zonae intact oocytes with purified GB reduces, in a concentration-dependent manner, the number of sperm able to bind to the zonae. The function of the soluble enzyme is not known. Its ability to bind both trypsin inhibitors and ZP3 suggests a possible role in gamete recognition.

GLQ223: an inhibitor of human immunodeficiency virus replication in acutely and chronically infected cells of lymphocyte and mononuclear phagocyte lineage.

GLQ223 is a highly purified, formulated preparation of trichosanthin, a 26-kDa plant-derived ribosome-inactivating protein with potent inhibitory activity against human immunodeficiency virus (HIV) in vitro. The compound produced concentration-dependent inhibition of HIV replication in acutely infected cultures of T-lymphoblastoid cells (VB cell line). Treatment with GLQ223 selectively reduced levels of detectable viral proteins compared to total cellular protein synthesis and produced a selective decrease in levels of viral RNA relative to total cellular RNA in acutely infected cells. Substantial inhibition of viral replication was observed at concentrations of GLQ223 that showed little inhibition of parallel uninfected cultures. Selective anti-HIV activity was also observed in cultures of primary monocyte/macrophages chronically infected with HIV in vitro. When freshly drawn blood samples from HIV-infected patients were treated with a single 3-hr exposure to GLQ223. HIV replication was blocked for at least 5 days in subsequently cultured monocyte/macrophages, without further treatment. The anti-HIV activity of GLQ223 in both acutely and chronically infected cells and its activity in cells of both lymphoid and mononuclear phagocytic lineage make it an interesting candidate as a potential therapeutic agent in HIV infection and AIDS.