Measurements of an ablator-gas atomic mix in indirectly driven implosions at the National Ignition Facility.

We present the first results from an experimental campaign to measure the atomic ablator-gas mix in the deceleration phase of gas-filled capsule implosions on the National Ignition Facility. Plastic capsules containing CD layers were filled with tritium gas; as the reactants are initially separated, DT fusion yield provides a direct measure of the atomic mix of ablator into the hot spot gas. Capsules were imploded with x rays generated in hohlraums with peak radiation temperatures of ∼294 eV. While the TT fusion reaction probes conditions in the central part (core) of the implosion hot spot, the DT reaction probes a mixed region on the outer part of the hot spot near the ablator-hot-spot interface. Experimental data were used to develop and validate the atomic-mix model used in two-dimensional simulations.

In vitro and in vivo stability of diluted recombinant factor VIII for continuous infusion use in haemophilia A.

Factor VIII (FVIII) replacement by continuous infusion (CI) is used postoperatively or after significant bleeding. For young paediatric patients, CI may require FVIII dilution. Variable stabilities of diluted full-length recombinant FVIII Kogenate FS (KG-FS) have been reported under different storage conditions. We investigated the recovery and stability of diluted KG-FS in vitro and in vivo. Kogenate FS was diluted to 50-120 U mL(-1) and its recovery and stability in glass vials or polypropylene syringes was determined. Furthermore, stability of KG-FS diluted to 80 U mL(-1)'administered' via single- and double-pump mock CI systems was tested. Finally, the in vivo stability of KG-FS diluted to approximately 60 U mL(-1) and administered postsurgically by CI with the double-pump to a paediatric patient with severe haemophilia A undergoing implantable venous access device placement was investigated. Initial KG-FS dilution resulted in a 10-20% FVIII loss; a further 25-30% loss occurred over 72 h in vials or syringes. With the double-pump, 1 h recovery was 35%, increasing to 80% by 24 h; the initial losses were because of the Y-infusion of a 10-fold larger volume of saline concomitantly with the FVIII. In vivo, CI resulted in stable FVIII activity levels within the target range. These in vitro results are important for the generation of CI guidelines for diluted KG-FS in the paediatric haemophilic population. That FVIII losses occur upon dilution and with the double-pump does not preclude use of diluted KG-FS. Indeed, stable FVIII levels were maintained when diluted KG-FS was administered by CI with the double-pump to a paediatric patient postsurgically.

Regulation of interferon-stimulated genes in peripheral blood leukocytes in pregnant and bred, nonpregnant dairy cows.

In ruminants, pregnancy results in up-regulation of a large number of IFN-stimulated genes (ISG) in the uterus. Recently, one of these genes was also shown to increase in peripheral blood leukocytes (PBL) during early pregnancy in sheep. Our working hypothesis is that conceptus signaling activates maternal gene expression in PBL in dairy cattle. The objectives of this study were to characterize ISG expression in PBL from pregnant (n = 20) and bred, nonpregnant (n = 30) dairy cows. Steady-state levels of mRNA for Mx1, Mx2, beta2-microglobulin, ISG-15, IFN regulatory factor-1, and IFN regulatory factor-2 were quantified. Holstein cows were synchronized to estrus and artificially inseminated (d 0). Blood samples were collected (coccygeal venipuncture) on d 0 and 16, 18, and 20 d after insemination for progesterone analysis and PBL isolation. Pregnancy was confirmed by transrectal ultrasonography at approximately 40 d after breeding. A status x day interaction was detected for Mx1, Mx2, and ISG-15 gene expression. When analyzed within day, levels of mRNA for ISG-15 and Mx1 were greater in pregnant compared with bred, nonpregnant cows on d 18 and 20, respectively. Expression of the Mx2 gene increased in the pregnant group compared with bred, nonpregnant cows on d 16, 18, and 20 after insemination. beta2-Microglobulin, IFN regulatory factor-1, and IFN regulatory factor-2 were not different between groups. The results clearly indicated that components of the innate immune response are activated in PBL during the period of pregnancy recognition and early embryo signaling. The physiological implications of these changes on maternal immune function are as yet unknown; however, they do provide a unique opportunity to identify bred, nonpregnant, cows 18 d after insemination in dairy cattle.

Nickel accumulation and nickel oxalate precipitation by Aspergillus niger.

A strain of Aspergillus niger isolated from a metal-contaminated soil was able to grow in the presence of cadmium, chromium, cobalt, copper, and unusually high levels of nickel on solid (8.0 mM) and in liquid (6.5 mM) media. This fungus removed >98% of the nickel from liquid medium after 100 h of growth but did not remove the other metals, as determined by inductively coupled plasma spectroscopy. Experiments with non-growing, live fungal biomass showed that nickel removal was not due to biosorption alone, as little nickel was bound to the biomass at the pH values tested. Furthermore, when the protonophore carbonyl cyanide p-(trifluoremetoxy) phenyl hydrazone (FCCP) was added to the actively growing fungus nickel removal was inhibited, supporting the hypothesis that energy metabolism is essential for metal removal. Analytical electron microscopy of thin-sectioned fungal biomass revealed that metal removed from the broth was localized in the form of small rectangular crystals associated with the cell walls and also inside the cell. X-ray and electron diffraction analysis showed that these crystals were nickel oxalate dihydrate.

Analysis of an engineered sulfate reduction pathway and cadmium precipitation on the cell surface.

We previously have genetically engineered an aerobic sulfate reduction pathway in Escherichia coli for the generation of hydrogen sulfide and demonstrated the pathway's utility in the precipitation of cadmium. To engineer the pathway, the assimilatory sulfate reduction pathway was modified so that cysteine was overproduced. Excess cysteine was then converted by cysteine desulfhydrase to an abundance of hydrogen sulfide, which then reacted with aqueous cadmium to form cadmium sulfide. In this study, observations of various E. coli clones were combined with an analysis of kinetic and transport phenomena. This analysis revealed that cysteine production is the rate-limiting step in the engineered pathway and provided an explanation for the phenomenon of cell surface precipitation. An analytical model showed that cadmium sulfide must form at the cell surface because the rate of cadmium sulfide formation is extremely fast and the rate of sulfide transport is relatively slow.

Aerobic sulfide production and cadmium precipitation by Escherichia coli expressing the Treponema denticola cysteine desulfhydrase gene.

The cysteine desulfhydrase gene of Treponema denticola was over-expressed in Escherichia coli to produce sulfide under aerobic conditions and to precipitate metal sulfide complexes on the cell wall. When grown in a defined salts medium supplemented with cadmium and cysteine, E. coli producing cysteine desulfhydrase secreted sulfide and removed nearly all of the cadmium from solution after 48 h. A control strain produced significantly less sulfide and removed significantly less cadmium. Measurement of acid-labile sulfide and energy dispersive X-ray spectroscopy indicated that cadmium was precipitated as cadmium sulfide. Without supplemental cysteine, both the E. coli producing cysteine desulfhydrase and the control E. coli demonstrated minimal cadmium removal.

Chemical denaturation and elevated folding temperatures are required for wild-type activity and stability of recombinant Methanococcus jannaschii 20S proteasome.

The 20S proteasome from the extreme thermophile Methanococcus jannaschii (Mj) was purified and sequenced to facilitate production of the recombinant proteasome in E. coli. The recombinant proteasome remained in solution at a purity level of 80-85% (according to SDS PAGE) following incubation of cell lysates at 70 degrees C. Temperature-activity profiles indicated that the temperature optima of the wild-type and recombinant enzymes differed substantially, with optimal activities occurring at 119 degrees C and 95 degrees C, respectively. To ameliorate this discrepancy, two recombinant enzyme preparations were produced, each of which included denaturation of the proteasome by 4 M urea followed by high-temperature (85 degrees C) dialysis. The wild-type temperature optimum was restored, but only if proteasome subunits were denatured and refolded prior to assembly (a preparation designated as alpha & beta). In contrast, when proteasome assembly preceded denaturation (designated alpha + beta) the optimum temperature was raised to a lesser degree. Moreover, the alpha & beta and alpha + beta preparations had apparent thermal half-lives at 114 degrees C of 54.2 and 26.2 min, respectively, and the thermostability of the less stable enzyme was more sensitive to a reduction in pH. Attainment of wild-type activity and stability thus required the proper folding of both the alpha- and beta-subunits prior to proteasome assembly. Consistent with this behavior, dual-scanning calorimetry (DSC) measurements revealed differences in the reassembly efficiency of the two proteasome preparations. The ability to produce structural conformers with dramatically different thermal optima and thermostabilities may facilitate the determination of molecular forces and structural motifs responsible for enzyme thermostablity and high-temperature activity.

Parallel synthesis and biocatalytic amplification of a cross-conjugated cyclopentenone library.

A combination of parallel chemical synthesis and biocatalysis has been used to prepare and amplify a library of cross-conjugated cyclopentenones. A number of marine and terrestrial natural products with antibiotic activity are known to incorporate this pharmacophore. The library was screened for anticancer, antimycobacterial, antifungal, and antibacterial activity. The positive results from the screens provide an indication of the structural features that are associated with activity in the various assays and suggest promising avenues for further inquiry.

Probing stability-activity relationships in the thermophilic proteasome from Thermoplasma acidophilum by random mutagenesis.

Structural perturbations (L65H, V12L/M27T, F35V) generated by random mutation of the beta-subunit were used to probe the relationship between stability and activity in the thermophilic proteasome from Thermoplasma acidophilum. The optimum temperature for activity of each mutant (approximately 95 degrees C) remained unchanged; however, each mutant was significantly less stable than the wild type. Stability, therefore, is not the factor limiting high-temperature activity. Interestingly, mutation L65H drastically reduced stability without affecting specific activity over a wide temperature range, providing evidence that activity and stability can be decoupled. To investigate the nature of the flexibility introduced by mutation, stability of the proteasome was examined under pressure. The application of 10,000 psi stabilized the wild-type proteasome 3.4 fold at 97 degrees C. When inactivation temperatures were chosen such that the rate of inactivation of the mutants was similar to that of the wild type, mutants with changes at the intersubunit interfaces (L65H and V12L/M27T) were similarly stabilized. Pressure was less effective in stabilizing mutant F35V, however, in which the substitution may have introduced a new pathway for inactivation.

Torsades de pointes associated with high dose levomethadyl acetate (ORLAAM).

A patient undergoing management of heroin dependency with high dosages of the long-acting methadone derivative, levomethadyl acetate HCl (LAAM; ORLAAM) developed a prolonged QTc interval and polymorphic QRS complexes on EKG consistent with torsades de pointes (TdP). The patient was taking other drugs known to prolong the QTc interval (fluoxetine and IV cocaine), and those known to antagonize the activity of the P450 enzymes responsible for the metabolism of LAAM and its active metabolite (fluoxetine, cocaine and marijuana). No previous reports have appeared in the literature attributing this adverse event to LAAM therapy; however, five similar cases have been reported to the manufacturer. Animal studies indicate that LAAM and metabolites prolong the action potential duration of myocardial cells. We propose that predisposed patients on high doses of LAAM may be at risk for developing TdP. Patients being treated with LAAM should receive dosages consistent with guidelines and be evaluated for concomitant diseases, interacting drug therapies, and EKG abnormalities.

Metabolic engineering of an aerobic sulfate reduction pathway and its application to precipitation of cadmium on the cell surface.

The conversion of sulfate to an excess of free sulfide requires stringent reductive conditions. Dissimilatory sulfate reduction is used in nature by sulfate-reducing bacteria for respiration and results in the conversion of sulfate to sulfide. However, this dissimilatory sulfate reduction pathway is inhibited by oxygen and is thus limited to anaerobic environments. As an alternative, we have metabolically engineered a novel aerobic sulfate reduction pathway for the secretion of sulfides. The assimilatory sulfate reduction pathway was redirected to overproduce cysteine, and excess cysteine was converted to sulfide by cysteine desulfhydrase. As a potential application for this pathway, a bacterium was engineered with this pathway and was used to aerobically precipitate cadmium as cadmium sulfide, which was deposited on the cell surface. To maximize sulfide production and cadmium precipitation, the production of cysteine desulfhydrase was modulated to achieve an optimal balance between the production and degradation of cysteine.

Engineering hydrogen sulfide production and cadmium removal by expression of the thiosulfate reductase gene (phsABC) from Salmonella enterica serovar typhimurium in Escherichia coli.

The thiosulfate reductase gene (phsABC) from Salmonella enterica serovar Typhimurium was expressed in Escherichia coli to overproduce hydrogen sulfide from thiosulfate for heavy metal removal (or precipitation). A 5.1-kb DNA fragment containing phsABC was inserted into the pMB1-based, high-copy, isopropyl-beta-D-thiogalactopyranoside-inducible expression vector pTrc99A and the RK2-based, medium-copy, m-toluate-inducible expression vector pJB866, resulting in plasmids pSB74 and pSB77. A 3. 7-kb DNA fragment, excluding putative promoter and regulatory regions, was inserted into the same vectors, making plasmids pSB103 and pSB107. E. coli DH5alpha strains harboring the phsABC constructs showed higher thiosulfate reductase activity and produced significantly more sulfide than the control strains under both aerobic and anaerobic conditions. Among the four phsABC constructs, E. coli DH5alpha (pSB74) produced thiosulfate reductase at the highest level and removed the most cadmium from solution under anaerobic conditions: 98% of all concentrations up to 150 microM and 91% of 200 microM. In contrast, a negative control did not produce any measurable sulfide and removed very little cadmium from solution. Energy-dispersive X-ray spectroscopy revealed that the metal removed from solution precipitated as a complex of cadmium and sulfur, most likely cadmium sulfide.

Posterior capsule opacification.

Posterior capsule opacification (PCO) is the most common complication following primary cataract surgery. Advances in intraocular lens (IOL) designs that have reduced the amount of PCO following surgery have been made. The understanding of how the IOL design effects PCO has also advanced. Lenses that provide a mechanical barrier between it and the posterior lens capsule seem to inhibit PCO to a greater degree. Intracapsular rings are now being explored to test and enhance this barrier effect. Major advances in the elimination of lens epithelial cells at the time of surgery especially by pharmacologic means have also been made. An immunotoxin specific for human lens epithelial cells shows promise and is under latter phase clinical development.

Pressure-induced thermostabilization of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus.

In this paper, elevated pressures up to 750 atm (1 atm = 101 kPa) were found to have a strong stabilizing effect on two extremely thermophilic glutamate dehydrogenases (GDHs): the native enzyme from the hyperthermophile Pyrococcus furiosus (Pf), and a recombinant GDH mutant containing an extra tetrapeptide at the C-terminus (rGDHt). The presence of the tetrapeptide greatly destabilized the recombinant mutant at ambient pressure; however, the destabilizing effect was largely reversed by the application of pressure. Electron spin resonance (ESR) spectroscopy of a spin-label attached to the terminal cysteine of rGDHt revealed a high degree of mobility, suggesting that destabilization is due to weakened intersubunit ion-pair interactions induced by thermal fluctuations of the tetrapeptide. For both enzymes, the stabilizing effect of pressure increased with temperature as well as pressure, reaching 36-fold for rGDHt at 105 degrees C and 750 atm, the largest pressure-induced thermostabilization of an enzyme reported to date. Stabilization of both native GDH and rGDHt was also achieved by adding glycerol. Based on the kinetics of thermal inactivation and the known effects of glycerol on protein structure, a mechanism of pressure-induced thermostabilization is proposed.

Inhibition of posterior capsule opacification with an immunotoxin specific for lens epithelial cells: 24 month clinical results.

PURPOSE: To assess the safety and effectiveness of an immunotoxin, MDX-RA, designed to inhibit posterior capsule opacification (PCO). SETTING: Eleven private practices in the United States. METHODS: This study comprised 63 eyes of 63 patients having extracapsular cataract extraction by phacoemulsification; these patients were enrolled in a Phase I/II clinical investigation of the immunotoxin MDX-RA. At the close of surgery, 21 patients were treated with placebo, 23 patients with 50 units of the immunotoxin, and 19 patients with 175 units of the immunotoxin as an aqueous solution. The patients were monitored for 24 months after primary cataract surgery using external eye and slitlamp examinations, visual acuity assessment, ophthalmoscopy, pachymetry, tonometry, endothelial cell counts, and lens capsule photography. Posterior capsule opacification, recorded on lens capsule photographs, was graded independently by a committee of 3 cataract surgeons. The incidence of neodymium:YAG (Nd:YAG) capsulotomy was projected from the opacification results. RESULTS: The immunotoxin, at the 50 unit dose, was well tolerated and effective in inhibiting PCO. At the 175 unit dose, there was a trend toward increased postoperative inflammation that was transient with no residua. From 6 to 24 months postoperatively, the 50 unit dose significantly inhibited PCO compared with the placebo (P < .05). This significant reduction in PCO translated into a significantly lower projected need for Nd:YAG capsulotomy in the 50 unit than the placebo group (P < .004). About 60% in the placebo group and 4% in the 50 unit group were projected to need an Nd:YAG capsulotomy by 3 years postoperatively. CONCLUSION: The immunotoxin was well tolerated and was effective in reducing PCO for up to 24 months after cataract surgery. Although these preliminary results are encouraging, a larger study is underway to determine whether the reduction in PCO by the immunotoxin decreases the need for Nd:YAG capsulotomy.

Mathematical model to predict the need for neodymium: YAG capsulotomy based on posterior capsule opacification rate.

PURPOSE: To evaluate a model to project the estimated time required before patients having primary phacoemulsification require neodymium:YAG (Nd:YAG) laser capsulotomy. SETTING: Eleven private practices in the United States. METHODS: Projections of time to capsulotomy were based on assessment of the early development of posterior capsule opacification (PCO) over time. The PCO data were collected during a clinical study to evaluate MDX-RA, an investigational immunotoxin designed to limit epithelial cell growth, preventing postsurgical PCO. From the PCO data, the estimated time to Nd:YAG capsulotomy in a placebo-treated group was compared with the actual time to capsulotomy in a cohort of patients from general practice who had had phacoemulsification. RESULTS: By 6 months, the mean Opacification Index in the MDX-RA group was significantly lower than that in the placebo group (P < .05) and it remained significantly lower at 12 (P < .001), 18 (P < .001), and 24 (P < .016) months. The rate of PCO in the MDX-RA group was approximately 6 times lower than that in the placebo group (P < .0004). Fifty-seven percent in the placebo group and 4% in the MDX-RA group were projected to require an Nd:YAG capsulotomy within 3 years of primary cataract surgery. Projected values for the placebo group were similar to actual values observed in the population-based cohort. CONCLUSIONS: This technique could be used to predict the need for Nd:YAG capsulotomy using early measurements of PCO.

Purification and functional characterization of a chaperone from Methanococcus jannaschii.

A chaperone from Methanococcus jannaschii has been purified to homogeneity with a single chromatographic step. The chaperone was identified and characterized using activity assays for characteristic chaperone abilities. The M. jannaschii chaperone binds unfolded proteins, protects proteins against heat-induced aggregation, and has a strongly temperature dependent ATPase activity. The chaperone has also been shown to inhibit the spontaneous refolding of a mesophilic protein at low temperatures. The purified chaperone complex has a M(r) of about 1,000,000 and consists of a single type of subunit with an approximate M(r) of 60,000. Analysis of partial sequence data reveals that this chaperone is the predicted protein product of the previously identified chaperonin gene in M. jannaschii (BULT et al., 1996). To our knowledge, this is the first functional characterization of a chaperone from a methanogen.

Cadmium removal by a new strain of Pseudomonas aeruginosa in aerobic culture.

A fluorescent pseudomonad (strain CW-96-1) isolated from a deep-sea vent sample grew at 30 degrees C under aerobic conditions in an artificial seawater medium and tolerated cadmium concentrations up to 5 mM. After 140 h, strain CW-96-1 removed > 99% of the cadmium from solution. Energy dispersive microanalysis revealed that the cadmium was removed by precipitation on the cell wall; sulfide production was confirmed by growth on Kligler's agar. Based on 16S ribosomal DNA sequencing and fatty acid analysis, the microorganism is closely related to Pseudomonas aeruginosa.

Pressure-enhanced activity and stability of a hyperthermophilic protease from a deep-sea methanogen.

We describe the properties of a hyperthermophilic, barophilic protease from Methanococcus jannaschii, an extremely thermophilic deep-sea methanogen. This enzyme is the first protease to be isolated from an organism adapted to a high-pressure-high-temperature environment. The partially purified enzyme has a molecular mass of 29 kDa and a narrow substrate specificity with strong preference for leucine at the P1 site of polypeptide substrates. Enzyme activity increased up to 116(deg)C and was measured up to 130(deg)C, one of the highest temperatures reported for the function of any enzyme. In addition, enzyme activity and thermostability increased with pressure: raising the pressure to 500 atm increased the reaction rate at 125(deg)C 3.4-fold and the thermostability 2.7-fold. Spin labeling of the active-site serine revealed that the active-site geometry of the M. jannaschii protease is not grossly different from that of several mesophilic proteases; however, the active-site structure may be relatively rigid at moderate temperatures. The barophilic and thermophilic behavior of the enzyme is consistent with the barophilic growth of M. jannaschii observed previously (J. F. Miller et al., Appl. Environ. Microbiol. 54:3039-3042, 1988).

Transient responses of hybridoma cells in continuous culture to step changes in amino acid and vitamin concentrations.

The effects of step-change increase in the concentrations of amino acids and vitamins on the metabolism, growth, and antibody productivity of a murine hybridoma cell line grown in continuous culture on serum-free medium are presented. Additions of the amino acids cysteine with methionine, tryptophan, and isoleucine with valine and vitamin B(12) (as cyanocobalamin) resulted in significant increases in viable cell concentrations. Additions of aspartate with asparagine, and threonine with vitamin B(1) (as thiamine hydrochloride) resulted in significant increases in final antibody concentrations. Substantial decrease in the fraction of amino acid nitrogen excreted as ammonia occurred upon supplementation with three times the normal concentrations of branched chain amino acids. Decreases in the fraction of amino acid nitrogen converted to ammonia were paralleled by increases in the fraction converted to alanine.