Solid-phase synthesis of ragweed allergen Ra5

A two-stage strategy was used to synthesize the 45-residue, four-disulfide-bonded ragweed protein allergen Ra5: (i) the protected linear chain was prepared by solid-phase, stepwise synthesis according to the sequence of the fully reduced protein; (ii) after removal from the resin and deprotection by hydrogen fluoride, the reduced chain was purified by gel filtration on Sephadex G-50 and allowed to fold “spontaneously” in the presence of oxidized dithiothreitol to form the disulfide links. The product (synthetic Ra5) was purified to disc-electrophoretic homogeneity by cycling through Sephadex G-50 and CM-Sephadex (overall yield: 6% theoretical). Synthetic Ra5 was closely similar in structure to the natural protein by amino acid analysis. polyacrylamide gel electrophoresis, tryptic peptide mapping, and circular dichroism spectra, as well asin vitro andin vivo immunoassays of antigenic/allergenic activities.     

Intracellular sequestration of 223Ra by the iron-storage protein ferritin

Incorporation of bone-seeking, alpha-particle-emitting, heavy-metal radionuclides dramatically increases the incidence of osteosarcoma in humans and experimental animals. The accumulation of these radionuclides within the mineral phase of the bone matrix is believed to result in local irradiation of only those proliferating cells close to the bone surface. We now present evidence for a more general pathway for the irradiation of target cells, mediated through the sequestration of heavy-metal radionuclides by the intracellular iron-storage protein ferritin. In vitro studies reveal the transfer of radionuclide from a 223Ra-transferrin complex into immunoprecipitable cytosolic ferritin. In vivo studies confirm the co-localization of incorporated 224Ra and cellular iron stores. This pathway would result in the highly localized irradiation of ferritin-containing cells. Since osteoblastic cells express large quantities of a ferritin isoform specialized in long-term metal storage, we suggest that this may represent an unrecognized source of intracellular irradiation by alpha-particle-emitting radionuclides. Such a local concentration within target cells has implications both for cellular dosimetry and for inferences of track length and target cell populations within the skeleton.     

The dosimetry of 224Ra in mouse bone

Calculations are described, based on experimental findings, which show the variation of absorbed dose from 224Ra in bone marrow of CBA/H mice. These calculations indicate that, following an injection of a leukaemogenic amount of 16 kBq 224Ra into these mice, most marrow cells in the cancellous bone of femur ends are killed but most marrow cells in the femur shaft survive. The calculations also suggest that the mean leukaemogenic absorbed dose of about 1.5 Gy is received by a population of marrow cells about 30 microns from bone surface in the femur shaft.     

Background concentration of 226Ra in terrestrial animals

Evidence that environmental levels of vanadium are increasing hasraised concern over the injection of vanadium into the environmentfrom anthropogenic sources. Two simple global mass balance models(simulating current and pre-industrial conditions) were developed todemonstrate the influence of anthropogenic vanadium on the globaldistribution of this trace metal. Current vanadium emissions owing toman's current industrial activities were estimated to comprise 30% oftotal atmosphere loading, 3% of total ocean loading, and 6% of totalland loading. These loadings were always considerably less than thoseresulting from non-anthropogenic sources or events. Differences notedbetween the pre-industrial and current models were not sufficientlygreat to suggest that injection of anthropogenic vanadium constitutes asignificant environmental threat on a global scale.     

Microbial release of 226Ra2+ from (Ba,Ra)SO4 sludges from uranium mine wastes

226Ra2+ is removed from uranium mine effluents by coprecipitation with BaSO4. (Ba,Ra)SO4 sludge samples from two Canadian mine sites were found to contain active heterotrophic populations of aerobic, anaerobic, denitrifying, and sulfate-reducing bacteria. Under laboratory conditions, sulfate reduction occurred in batch cultures when carbon sources such as acetate, glucose, glycollate, lactate, or pyruvate were added to samples of (Ba,Ra)SO4 sludge. No external sources of nitrogen or phosphate were required for this activity. Further studies with lactate supplementation showed that once the soluble SO4(2-) in the overlying water was depleted, Ba2+ and 226Ra2+ were dissolved from the (Ba,Ra)SO4 sludge, with the concurrent production of S2-. Levels of dissolved 226Ra2+ reached approximately 400 Bq/liter after 10 weeks of incubation. Results suggest that the ultimate disposal of these sludges must maintain conditions to minimize the activity of the indigenous sulfate-reducing bacteria to ensure that unacceptably high levels of 226Ra2+ are not released to the environment.     

226Ra induced bone-cancers: the effects of a delayed Na-alginate treatment

At the present time no unequivocal evidence exists which shows that a reduction in the body-burden of a radionuclide by decorporative treatment results in a proportional decrease in the risk of long-term radiation effects. We have investigated the effectiveness of the daily administration of Na-alginate via the diet in removing 226Ra from the skeleton and in reducing the number of late effects such as osteosarcomas. The animals used were male C57Bl mice which had been injected with one of three different amounts of 226Ra (4.4, 10.7 or 24.8 kBq) four days prior to the onset of the decorporative treatment. The results showed that although this treatment was able to produce a substantial reduction in the 226Ra content of the mice it did not reduce the incidence of osteosarcoma. These results question the effectiveness of decorporation procedures initiated at longer times after contamination.     

Microbial release of 226Ra2+ from (Ba,Ra)SO4 sludges from uranium mine wastes.

226Ra2+ is removed from uranium mine effluents by coprecipitation with BaSO4. (Ba,Ra)SO4 sludge samples from two Canadian mine sites were found to contain active heterotrophic populations of aerobic, anaerobic, denitrifying, and sulfate-reducing bacteria. Under laboratory conditions, sulfate reduction occurred in batch cultures when carbon sources such as acetate, glucose, glycollate, lactate, or pyruvate were added to samples of (Ba,Ra)SO4 sludge. No external sources of nitrogen or phosphate were required for this activity. Further studies with lactate supplementation showed that once the soluble SO4(2-) in the overlying water was depleted, Ba2+ and 226Ra2+ were dissolved from the (Ba,Ra)SO4 sludge, with the concurrent production of S2-. Levels of dissolved 226Ra2+ reached approximately 400 Bq/liter after 10 weeks of incubation. Results suggest that the ultimate disposal of these sludges must maintain conditions to minimize the activity of the indigenous sulfate-reducing bacteria to ensure that unacceptably high levels of 226Ra2+ are not released to the environment.     

Microdistribution and local dosimetry of 226Ra in trabecular bone of the beagle

Sections of lumbar vertebral bodies of young adult beagle dogs have been analyzed autoradiographically to characterize and quantify the local distribution of 226Ra by means of a scanning microscope photometer. The animals received a single injection of 355 kBq/kg body weight and were serially sacrificed at 5 to 1381 days postinjection. Hotspot concentrations decreased from about 51 kBq/g bone at 5 days to 20 kBq/g at 1381 days postinjection. The diffuse concentration changed from 8.3 to 1.9 kBq/g. The mean 226Ra concentration in the trabecular areas scanned was initially higher and at the end of the observation period lower than the average calculated for the whole lumbar vertebral column. Density and area of, and fraction of bone activity in, hotspots virtually remained constant. With time hotspots tended to become translocated into bone volume. Mean dose rates to lining cells from both hotspots and diffuse labels decreased from about 210 mGy/d at early postinjection times to 105 mGy/d. This corresponds to 2.5 to 1.1 times the average skeletal dose rate. A discussion of the level of irradiation in terms of hit frequencies shows that osteoblasts in the initial phase of hotspot formation receive about 60 hits to their nucleus for the duration of bone formation. After about 6 months, however, the 226Ra concentration in new bone and the corresponding hit frequency appears to be low enough that interference with bone formation is unlikely. Morphometric measurements showed that abnormal bone accretion and thickening of trabeculae occurred. This was interpreted as an imbalance between bone formation and resorption. Both formation and resorption seem to be substantially lowered compared to control animals.     

Polymorphism of Crystals of Salmonella minnesota Re and Ra Lipopolysaccharides

Salmonella minnesota Re and Ra lipopolysaccharides (LPSs) formed three-dimensional crystals when they were precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl₂ and incubated in 70% ethanol containing 250 mM MgCl₂ at 4 C. Besides typical shapes of crystals, hexagonal plates and solid columns, which were already reported (J. Bacteriol. 172: 1516–1528 (1990)), the LPSs thus treated formed crystals possessing various shapes such as square or rectangular plate, lozenge plate, discoid, and truncated hexangular pyramid forms. Electron diffraction patterns from all these crystals except square or rectangular plate crystals obtained by electron irradiation from the direction perpendicular to the basal plane were essentially the same as those from hexagonal plate crystals, indicating that they consist of hexagonal lattices with the lattice constant of 4.62 Å. From these results as well as the results of electron microscopic observations of these crystals, it was concluded that all these crystals except square or rectangular plate crystals are composed of hexagonal plate sheets as the basic structural units. Square or rectangular crystals were assumed to correspond to the {1011} planes of solid hexagonal column crystals.     

Chemical Composition of the Cell Wall of the H37Ra Strain of Mycobacterium tuberculosis

The cell wall of the H37Ra strain of Mycobacterium tuberculosis was isolated and freed of extraneous noncovalently linked material by a series of extraction and enzymatic procedures. Chemical analysis of the cell wall has revealed the following composition: 22.8% amino acids, principally alanine, glutamate, and diaminopimelate in a molar ratio of 1:1.8:0.8; 24.7% reducing sugars, all in the form of arabinose and galactose in a molar ratio of 2.6:1; and 3.95% amino sugars, all in the form of glucosamine, muramic acid, and galactosamine in a molar ratio of 1:6.6:0.8. About 32.1% of the dry weight of the cell wall is lipid, of this about 55% is in the form of two series of mycolic acids. Each series of mycolic acids contains two homologues differing by 28 mass units. One pair of homologues contains in each a carbonyl function and an unsaturated double bond; the other pair contains two cyclopropane groups in each homologue. The remaining lipids are composed principally of normal saturated fatty acids, including tuberculostearic acid.     

Rabbit skeletal muscle acylphosphatase: the amino acid sequence of form Ra1

Acylphosphatase was purified from rabbit skeletal muscle by a procedure involving an affinity chromatography step on immunoadsorbent and subsequent ion-exchange chromatography. Three molecular forms with acylphosphatase activity, named Ra1, Ra2, and Ra3, were purified and characterized with respect to molecular weight, amino acid composition, and main kinetic parameters. The amino acid sequence of Ra1 is given in the present paper. The Ra1 form consists of a single polypeptide chain of 98 amino acid residues and contains only one cysteine residue at position 21 that is S-S bound to glutathione. The polypeptide chain has an acetyl group blocking the NH2 terminus. Ra1, Ra2, and Ra3 are compared with the corresponding molecular forms isolated from skeletal muscle of horse and turkey.     

Local distribution and dosimetry of 226Ra in the trabecular skeleton of the beagle.

Young adult beagle dogs received a single injection of 38.1 kBq/kg body wt 226Ra and were serially sacrificed at 4 to 2955 days postinjection. Samples of sites of trabecular bone in the lumbar vertebral body, proximal ulna, and distal femoral metaphysis and epiphysis were analyzed autoradiographically. The time-dependent changes in the average 226Ra concentrations in the four regions were analyzed in terms of a compartmental model. The clearance rate from the lumbar vertebral body was about four times more rapid than for the proximal ulna and distal femoral epiphysis. Ratios of hotspot to diffuse label concentrations varied from about 10 to 23. The dose rate to the endosteum ranged between 8.7 and 39.5 mGy/day initially and 4 and 10.5 mGy/day toward the end of the observation period. Mean marrow dose rates were lower by a factor of 3 to 9.5. During their residence time the nuclei of bone lining cells receive a maximum dose of 8 Gy in the proximal ulna (2955 days after injection) and a minimum dose of 0.63 Gy in the lumbar vertebra (2955 days after injection). This corresponds on the average to 17 and 1.4 alpha-particle hits to the cell nuclei, respectively.     

Immunochemical and genetic studies of Amb.t. V (Ra5G), an Ra5 homologue from giant ragweed pollen

Giant ragweed pollen allergen Amb.t. V (Ra5G), a homologue of short ragweed pollen Amb.a. V (Ra5S), was isolated in ultrapure form from a 16-min extract of ragweed pollen by a combination of molecular sieving through an Amicon hollow fiber cartridge (H1P5), cation-exchange chromatography, and gel filtration. The size was found to be 4400 daltons (D) by amino acid analysis and 6000 D by SDS-PAGE, and the pI was 8.3 as determined by isoelectric focusing. There was no cross-reactivity detected between the two Amb. V antigens by immunodiffusion and IEP with the use of hyperimmune antisera raised against crude or highly purified antigens. Cross-reactivity between the two Amb. V antigens was further investigated by inhibition double antibody radioimmunoassay by using the sera of nine selected ragweed-allergic patients who had recently been immunized with either mixed short-giant ragweed pollen extract or with short ragweed extract alone and who had IgG antibodies (Ab) to Amb.t. V and generally to Amb.a. V. Unlabeled Amb.t. V did not inhibit the binding of 125I-Amb.a. V to the IgG Ab in any of the sera tested. Conversely, unlabeled Amb.a. V produced some inhibition of the binding of 125I-Amb.t. V to the patients' IgG Ab, primarily in those patients who had received immunotherapy with short ragweed alone. This weak cross-reactivity was probably a result of the primary structural homology between the two protein allergens. The sera from two groups of ragweed-allergic individuals were investigated for the presence of IgG and IgE Ab to Amb.t. V. The presence of IgG Ab was found to be associated both with previous (or current) immunotherapy with giant ragweed extract and with HLA-Dw2. The HLA association is of interest in view of the previously established association between Dw2 and response toward the homologue Amb.a. V. The result suggests the existence of a similar genetic control at the primary level of antigenic recognition of the two Amb. V antigens.