First experiment with HELIOS: the structure of 13B.

A first experiment is reported that makes use of a new kind of spectrometer uniquely suited to the study of reactions with radioactive beams in inverse kinematics, the helical orbit spectrometer, HELIOS. The properties of some low-lying states in the neutron-rich N=8 nucleus 13B were studied with good resolution. From the measured angular distributions of the (d,p) reaction and the relative spectroscopic factors, spin and configuration assignments of the first- and third-excited states of this nucleus can be constrained.

Prospective randomized trial of azathioprine in cryopreserved valved allografts in children.

BACKGROUND: The purpose of this study was to prospectively assess the effects of azathioprine on the humoral immune response to HLA alloantigens and allograft function in children receiving cryopreserved valved allografts. METHODS: We randomized 13 children to receive azathioprine or not to receive azathioprine (controls) after receiving a cryopreserved valved allograft. Azathioprine patients received intraoperatively 4 mg/kg of azathioprine and 2.0 +/- 0.5 mg/kg once daily for 3 months after operation. Panel reactive antibodies against HLA class I and class II alloantigens were measured before, 1 month, and 3 months after operation. RESULTS: Panel reactive antibodies were not significantly different between the azathioprine and control groups before (0.0% +/- 0% versus 1.6% +/- 1%), 1 month (59% +/- 17% versus 71% +/- 12%), or 3 months (84% +/- 15% versus 96% +/- 1.3%) after operation. There were no differences in degree of allograft valve stenosis between azathioprine (31.5 +/- 26 mm Hg, 13.4 +/- 7 months postoperatively) and control groups (25.4 +/- 11 mm Hg, 17.2 +/- 10 months postoperatively) or allograft valve insufficiency. CONCLUSIONS: Azathioprine does not significantly decrease the immune response to HLA alloantigens or affect the function of cryopreserved valved allografts used in children to repair congenital heart defects.

Inhibition of human telomerase in immortal human cells leads to progressive telomere shortening and cell death.

The correlation between telomerase activity and human tumors has led to the hypothesis that tumor growth requires reactivation of telomerase and that telomerase inhibitors represent a class of chemotherapeutic agents. Herein, we examine the effects of inhibition of telomerase inside human cells. Peptide nucleic acid and 2'-O-MeRNA oligomers inhibit telomerase, leading to progressive telomere shortening and causing immortal human breast epithelial cells to undergo apoptosis with increasing frequency until no cells remain. Telomere shortening is reversible: if inhibitor addition is terminated, telomeres regain their initial lengths. Our results validate telomerase as a target for the discovery of anticancer drugs and supply general insights into the properties that successful agents will require regardless of chemical type. Chemically similar oligonucleotides are in clinical trials and have well characterized pharmacokinetics, making the inhibitors we describe practical lead compounds for testing for an antitelomerase chemotherapeutic strategy.

Detection of radon decay products in rainwater.

The Argonne National Laboratory-East (ANL-E) Environmental Radiation Monitoring System measures and records ambient radiation levels and provides detection capability for radon decay products in rain clouds. These decay products in rainwater tracked into a facility on the shoes of workers can cause false alarms from hand and shoe monitors. The monitors at ANL-E can easily detect the radon decay products, and the 19.6 and 26.8 min half-lives of the beta-particle emitters are long enough in many cases for sufficient activity to still be present to initiate a contamination alarm when the shoes are checked for radioactivity. The Environmental Radiation Monitoring System provides a warning when precipitation contains elevated levels of radon decay products. It is based on a prototype developed at the Super Collider Laboratory. During its first year of operation there were nine alarms from radon decay products with an alarm trigger point set at 30% greater than background. The alarms occurred at both monitoring stations, which are approximately 1,000 m apart, indicating large diameter radon clouds. The increases in background were associated with low atmospheric pressure. There was no correlation with radon released from the coal-burning steam plant on the site. Alarms also occurred when short-lived accelerator-produced radioactivity in the exhaust stack plume passed over the NaI(Tl) detector in one of the stations. The 450 MeV proton accelerator near the station produced 12C, 13N, and 15O by spallation of air nuclei. The gamma-ray spectrum from the plume from the accelerator exhaust stack was dominated by the 511 keV annihilation gamma rays from decay of these radionuclides. These gamma rays were easily distinguished from the 609 keV, 1,120 keV, and 1,764 keV gamma rays emitted by the radon decay products.

Substrate specificity of prostate-specific antigen (PSA).

BACKGROUND: The serine protease prostate-specific antigen (PSA) is a useful clinical marker for prostatic malignancy. PSA is a member of the kallikrein subgroup of the (chymo)trypsin serine protease family, but differs from the prototypical member of this subgroup, tissue kallikrein, in possessing a specificity more similar to that of chymotrypsin than trypsin. We report the use of two strategies, substrate phage display and iterative optimization of natural cleavage sites, to identify labile sequences for PSA cleavage. RESULTS: Iterative optimization and substrate phage display converged on the amino-acid sequence SS(Y/F)Y decreases S(G/S) as preferred subsite occupancy for PSA. These sequences were cleaved by PSA with catalytic efficiencies as high as 2200-3100 M-1 s-1, compared with values of 2-46 M-1 s-1 for peptides containing likely physiological target sequences of PSA from the protein semenogelin. Substrate residues that bind to secondary (non-S1) subsites have a critical role in defining labile substrates and can even cause otherwise disfavored amino acids to bind in the primary specificity (S1) pocket. CONCLUSION: The importance of secondary subsites in defining both the specificity and efficiency of cleavage suggests that substrate recognition by PSA is mediated by an extended binding site. Elucidation of preferred subsite occupancy allowed refinement of the structural model of PSA and should facilitate the development of more sensitive activity-based assays and the design of potent inhibitors.

A randomized, placebo-controlled trial of granulocyte colony-stimulating factor administration to newborn infants with neutropenia and clinical signs of early-onset sepsis.

OBJECTIVE: To determine whether recombinant human granulocyte colony-stimulating factor (G-CSF) administration: 1) accelerates production of neutrophils; 2) increases bone marrow stored and precursor neutrophils; and 3) is safe in newborn infants with neutropenia and clinical signs of early-onset sepsis. STUDY DESIGN: We randomized 20 infants with neutropenia and clinical signs of early-onset sepsis in the first 3 days of life to receive G-CSF (10 microg/kg/d) or placebo for 3 days. Entry criteria included neutropenia as defined by Manroe criteria, an elevated immature to total neutrophil ratio [(I/T) >/=0.25], and a requirement for ventilatory support. Cultures were obtained and antibiotics initiated on all study infants. Circulating absolute neutrophil count (ANC), I/T ratio, bone marrow neutrophil storage pool (NSP) and neutrophil proliferative pool (NPP), and plasma G-CSF concentrations were evaluated. Also, severity of illness as determined using the Score for Neonatal Acute Physiology (SNAP), morbidity, and mortality were recorded. RESULTS: Circulating ANC increased in both G-CSF and placebo recipients by day 1. Also, the I/T neutrophil ratio decreased in both G-CSF and placebo recipients. There were no significant differences in the ANC or I/T ratio between the two groups during the study period. Similarly, bone marrow NSP and NPP did not differ between G-CSF and placebo recipients at study entry or day 2. No differences were observed in the secondary outcome measures including severity of illness, morbidity, and mortality. CONCLUSIONS: Administration of recombinant G-CSF to infants with neutropenia and clinical signs of early-onset sepsis did not increase circulating ANC, or bone marrow NSP and NPP compared with placebo. No differences were observed between G-CSF and placebo recipients in severity of illness, morbidity, or mortality. No adverse effects of G-CSF administrations were noted.

Leaching of accelerator-produced radionuclides.

Leaching of radionuclides produced in soil and rock by high energy proton-induced radiation was studied for the SSC site. Comparison was made with predictions of a Monte Carlo code CASIM and previous results for the Fermilab site. The principal long-lived radionuclides were 3H and 22Na in agreement with Fermilab results. A few other radionuclides were present at lower concentrations in a subset of the samples. For example, 134Cs was detected in a few SSC water samples. Leaching from SSC chalk was dependent on previous weathering and on leaching time. The more soil-like marl and shale were leached more rapidly. Results of this study, in conjunction with the SSC groundwater model, show that adequate groundwater protection would have been maintained for an accidental loss of the entire proton beam at a point in the SSC Collider tunnel. Early warning techniques developed are directly applicable to soil activation monitoring at other facilities.

Groundwater activation at the Superconducting Super Collider: a new design model.

A groundwater activation model was developed for use in designing the accelerators at the Superconducting Super Collider Laboratory. This model is based on the concept of a 4-m-thick "activation zone" surrounding the accelerator enclosure, which contains over 99% of the soil activation caused by beam losses. Empirical shielding formulae based on computer simulations indicate that the soil activation in the activation zone decreases exponentially with distance from the tunnel enclosure. From this assumption, the average activation in the activation zone is derived. It is shown that the average activity concentration in the activation zone is equal to the activity concentration 1 m from the accelerator enclosure. The activation concentration in the water averaged over the volume of the activation zone is compared to the drinking water standards. The goal of this model is to meet the drinking water regulatory standards by averaging the activation in the activation zone. Groundwater activation concentrations have been calculated for the Super Collider utilizing experimental measurements of production cross sections and leachability factors. Comparison is made to the groundwater activation criterion for both routine and accidental beam losses.

Evidence for in vivo but not in vitro expression of a Borrelia burgdorferi outer surface protein F (OspF) homologue.

Protein export signals from the low-passage 297 strain of Borrelia burgdorferi were cloned as fusions with an Escherichia coli alkaline phosphatase (PhoA) reporter lacking a signal sequence. One PhoA+ clone (BbK2.10-PhoA) was derived from a borrelial lipoprotein. Although the polypeptide encoded by the full-length bbk2.10 gene had 76% similarity and 56% identity to outer surface protein F (OspF) from B. burgdorferi strain N40, antibodies directed against recombinant forms of the two proteins revealed that they were not cross-reactive. The nucleotide sequences of bbk2.10 and ospF from the N40 and 297 strains, respectively, were determined to confirm that the N40 and 297 strains each contained both genes. Southern blot analysis revealed that bbk2.10 is a single-copy gene and that the B. burgdorferi strain 297 and N40 genomes appeared to contain one other gene more closely related to ospF than bbk2.10. It was particularly noteworthy that ospF, but not bbk2.10, was expressed in vitro while B. burgdorferi-infected mice generated antibodies reactive with both lipoproteins. To help confirm that the BbK2.10-reactive antibodies produced by the B. burgdorferi-infected mice were specific for that protein, a second gene, bbk2.11, which hybridized with the ospF probe was cloned; the corresponding polypeptide reacted strongly with OspF antisera but failed to react with BbK2.10-specific antisera. Taken together, these data demonstrate that BbK2.10, BbK2.11, and OspF comprise a B. burgdorferi lipoprotein family and that at least one member (BbK2.10) appears to be expressed only during infection.

Chemiluminescent analysis of Borrelia burgdorferi penicillin-binding proteins using ampicillin conjugated to digoxigenin.

Knowledge of the penicillin-binding proteins (PBPs) of Borrelia burgdorferi is important for understanding both the targets of beta-lactams used therapeutically for Lyme borreliosis and the complex membrane biology of the distinctive spirochetal pathogen which causes Lyme disease. In this study, the PBPs of a number of B. burgdorferi strains and variants were examined using a rapid and sensitive chemiluminescent assay which employs ampicillin conjugated to digoxigenin (dig-amp). The minimum inhibitory concentration of dig-amp for B. burgdorferi high-passage strain B31 (0.012 micrograms/ml) was essentially no different from that of free ampicillin (0.025 micrograms/ml). Dig-amp bound specifically to B. burgdorferi B31 PBPs with molecular masses of 92, 80, 65, 46, 40, 34, 31, 29, 22, 20 and 13 kDa; the 31 kDa and 34 kDa PBPs were proven to be OspA and OspB, respectively. All of the borrelial PBPs were present in the cytoplasmic membrane fraction of B. burgdorferi, findings consistent with their activities as PBPs but inconsistent with OspA and OspB as surface-exposed outer membrane lipoproteins. Furthermore, among the PBP profiles of other high- and low-passage variants of B. burgdorferi strains Sh-2-82, HB19, and N40, which differed somewhat from one another, OspD (28 kDa) but not OspC (22-25 kDa) also was strongly implicated as a PBP; however, OspC possessed a gel mobility easily misconstrued as that of a 26 kDa PBP often expressed reciprocally with OspB. The ramifications of classifying OspA, OspB, and OspD as PBPs are discussed. While the current inability to genetically manipulate B. burgdorferi hinders determining which of the borrelial PBPs are essential for spirochetal viability (i.e., are the lethal targets of beta-lactams), a priori knowledge of the borrelial PBPs will facilitate the production and purification of recombinant derivatives whose activities can be assessed further in vitro.

Characterization of outer membranes isolated from Borrelia burgdorferi, the Lyme disease spirochete.

The lack of methods for isolating Borrelia burgdorferi outer membranes (OMs) has hindered efforts to characterize borrelial surface-exposed proteins. Here we isolated OMs by immersion of motile spirochetes in hypertonic sucrose followed by isopycnic ultracentrifugation of the plasmolyzed cells. The unilamellar vesicles thus obtained were shown to be OMs by the following criteria: (i) they contained OspA and OspB; (ii) they did not contain flagellin, NADH oxidase activity, or the 60-kDa heat shock protein; and (iii) their morphology by freeze-fracture electron microscopy was identical to that of OMs of intact organisms. Consistent with previous studies which employed immunoelectron microscopy and detergent-based solubilization of B. burgdorferi OMs, only small proportions of the total cellular content of OspA or OspB were OM associated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fluorography of OMs from spirochetes metabolically radiolabeled with [3H]palmitate or 35S-amino acids demonstrated that the OMs contained both nonlipidated and lipidated proteins. This fractionation procedure was also used to isolate OMs from virulent and avirulent isolates of the well-characterized B. burgdorferi N40 strain. SDS-PAGE fluorography revealed that OMs from the two isolates differed with respect to both nonlipoprotein and lipoprotein constituents. When whole cells, protoplasmic cylinders, and OMs were immunoblotted against sera from mice persistently infected with B. burgdorferi N40, the majority of antibody reactivity was directed against intracellular proteins. The availability of isolated OMs should facilitate efforts to elucidate the complex relationship(s) between B. burgdorferi membrane composition and Lyme disease pathogenesis.

Measurements of radioactive gaseous releases to air from target halls at a high-energy proton accelerator.

Measurements of induced radioactivity concentration in air were made at five target halls associated with the Fermilab high-energy proton synchrotron. A gas flow-through ionization chamber was used to continuously sample air released from an exhaust stack at each location. The radioactive decay of a grab sample of air from each target hall was recorded, and a best-fit of each decay curve was made via iterative steps to determine the mixture of radionuclides present. The radionuclides 11C, 13N, 15O, 38Cl, 39Cl, and 41Ar were identified. Two distinct radionuclide mixtures or "signatures" were found and were correlated to the geometry of the proton loss point. The normalized activity release rates for the five target areas agree within a factor of two of the average value of 2.7 muBq per proton for 800 GeV protons.

Dose calibrator readings due to radionuclidic impurities found in radiopharmaceuticals.

The influence of the radionuclidic impurities in radiopharmaceuticals on the reading of sample activities in a dose calibrator has been studied. As a practical example, commercially available 123I was used to measure the effect of radioactive impurities on "dose calibrator" readings. The impurities present in commercially available 123I were identified and quantified by the use of a Ge(Li) detector, and the response of a dose calibrator to each impurity was determined. The contribution of radioactive impurities to sample readings was determined for each sample of 123I studied. It was found that impurities may increase the dose calibrator readings up to 31%, even though the impurities were within the warranty given by the manufacturer.