The disposal of radioactive ferric floc.

An iron hydroxide floc is used as treatment for adsorbing low amounts of actinides during nuclear fuel re-processing. This waste is cemented only after pre-treatment with Ca(OH)(2). Characterisation of all simulant material has been undertaken using XRD, TGA and SEM/EDS. The floc is a moderately alkaline colloidal slurry containing approximately 15wt% solids, with the main particulate being an amorphous hydrated iron oxide. The main phase formed during pre-treatment appears to be an X-ray amorphous hydrated calcium-ferrate phase. Embedded within this are small amounts of crystalline Ca(OH)(2), calcite, Fe(6)(OH)(12)(CO(3)), Ca(6)Fe(2)(SO(4))(3)(OH)(12).26H(2)O and Ca(3)B(2)O(6), and can form depending on concentrations of Ca(OH)(2) and time. Apart from Ca(OH)(2) and calcite, none of the crystalline phases detected during pre-treatment are detected when the floc is encapsulated in an OPC/PFA composite cement hydrated for 90 days. The main crystalline phase detected in the hardened wasteform is a solid solution hydrogarnet, Ca(3)AlFe(SiO(4))(OH)(8), known as C(3)(A,F)SH(4) in cement chemistry nomenclature.

Concomitant changes in mitochondria and intermediate filaments during heat shock and recovery of chicken embryo fibroblasts.

Utilizing video-enhanced differential interference contrast microscopy of chicken embryo fibroblasts, we observed dramatic changes in the localization and morphology of mitochondria shortly after cells were subjected to a mild heat shock. At normal temperatures mitochondria were distributed in the cell cytoplasm as elongated, tubular, and dynamic organelles but upon heat shock they moved to the perinuclear region and formed a tight ring of short swollen and--in some cases--fused vesicles. Vital dye staining of mitochondria with rhodamine 123 and indirect immunofluorescence staining with antibodies against the mitochondrial-matrix protein, HSP 60, confirmed these results. Using cells double labeled with antibodies to vimentin and the HSP 60 protein, we found that the changes in mitochondria were accompanied by perturbations of the intermediate filament network that we and others have reported previously for heat shocked cells. Microtubules remained largely unaltered by our heat shock treatment and the redistribution of intermediate filaments and mitochondria occurred even in the presence of taxol, a microtubule stabilizing drug. The effects of heat shock on mitochondria were reversed when cells were returned to normal temperatures and their recovery to their normal state coincided with return of normal intermediate filament morphology. This recovery was blocked in cells treated with actinomycin D during heat shock, a result indicating that a heat shock protein may be required for recovery. These data are consistent with previously published observations that mitochondria are associated with the intermediate filament network but they extend this interaction to a cell system responding to a physiological stress normally experienced by the intact organism.

Inhibition of influenza virus formation by a peptide that corresponds to sequences in the cytoplasmic domain of the hemagglutinin.

A decapeptide with a sequence corresponding to the cytoplasmic domain of the influenza virus hemagglutinin inhibited the release of virus particles and infectious virions when added to infected cultured cells for a 2-hr period during a one-cycle growth. Inhibition was dose-dependent in the range of 50 to 250 micrograms/ml. The peptide did not affect formation of intracellular virus-specific proteins or assembly of nucleocapsids and did not inhibit replication of two unrelated enveloped RNA viruses, Sindbis virus and vesicular stomatitis virus. Peptides of similar size but different in sequence were ineffective. We postulate that this peptide acts as a competitive inhibitor for virus-specific protein-protein interactions between the hemagglutinin and the matrix protein or nucleocapsid during virus assembly. These data offer an approach to the development of antiviral drugs based on virus specific activities.

Ultrastructural and biochemical analysis of the stress granule in chicken embryo fibroblasts.

The ultrastructure and biochemical composition of cytoplasmic particles that form in chicken embryo fibroblasts during stress have been analyzed. We showed previously that these particles contained the small stress protein, sp 24, and antibodies specific to sp 24 were used here to identify the stress granule. In thin sections, the stress granule was a densely staining, membraneless, cytoplasmic body and appeared as a highly condensed area of cytoplasm in freeze-fracture preparations. Hypotonic swelling of cells before freeze-fracture analysis revealed a basketlike structure composed of interconnecting protein cables. No other proteins could be cross-linked to sp 24 when stress granules were treated with dithiobis-(succinimidyl propionate). High resolution autoradiographic analysis with [3H]uridine failed to identify any associated RNA synthesized in the period immediately before the stress. Thus the stress granule appears to be composed predominantly of sp 24 aggregates. Sp 24 could be purified to homogeneity from the stress granule by solubilization in 8 M urea and anion exchange chromatography.

The dynamic state of heat shock proteins in chicken embryo fibroblasts.

Subcellular fractionation and immunofluorescence microscopy have been used to study the intracellular distributions of the major heat shock proteins, hsp 89, hsp 70, and hsp 24, in chicken embryo fibroblasts stressed by heat shock, allowed to recover and then restressed. Hsp 89 was localized primarily to the cytoplasm except during the restress when a portion of this protein concentrated in the nuclear region. Under all conditions, hsp 89 was readily extracted from cells by detergent. During stress and restress, significant amounts of hsp 70 moved to the nucleus and became resistant to detergent extraction. Some of this hsp 70 was released from the insoluble form in an ATP-dependent reaction. Hsp 24 was confined to the cytoplasm and, during restress, aggregated to detergent-insoluble perinuclear phase-dense granules. These granules dissociated during recovery and hsp 24 could be solubilized by detergent. The nuclear hsps reappeared in the cytoplasm in cells allowed to recover at normal temperatures. Sodium arsenite also induces hsps and their distributions were similar to that observed after a heat shock, except for hsp 89, which remained cytoplasmic. We also examined by immunofluorescence the cytoskeletal systems of chicken embryo fibroblasts subjected to heat shock and found no gross morphological changes in cytoplasmic microfilaments or microtubules. However, the intermediate filament network was very sensitive and collapsed around the nucleus very shortly after a heat shock. The normal intermediate filament morphology reformed when cells were allowed to recover from the stress. Inclusion of actinomycin D during the heat shock--a condition that prevents synthesis of the hsps--did not affect the intermediate filament collapse, but recovery of the normal morphology did not occur. We suggest that an hsp(s) may aid in the formation of the intermediate filament network after stress.

Induction of heat-shock proteins in the embryonic chicken lens.

The 11-day embryonic chicken lens displays a rapid and vigorous response to a heat shock. Exposure of the lens to elevated temperature (45 degrees C) dramatically increased the synthesis of three proteins with subunit molecular weights of 89,000, 70,000 and 24,000. These lens heat-shock proteins are identical to the heat-shock proteins of cultured chicken embryo fibroblasts in electrophoretic mobility and immunological cross-reactivity. Maximum induction of lens heat-shock protein synthesis occurred upon incubation at 45 degrees C for about 1-1.5 hr and was blocked in the presence of actinomycin D. The functional half-lives at 37 degrees C of the mRNAs encoding the lens heat-shock proteins were about 3-5 hr. Synthesis of normal lens proteins continued throughout the heat shock, but delta-crystallin was unusual in that its synthesis initially rose upon heat shock, a behavior similar to that of a heat-shock protein. Proteins which co-migrated with each of the major lens heat-shock proteins were detected immunologically in non-stressed lens extracts. The endogenous lens protein, p24, which cross-reacted with antibodies raised against the small heat-shock protein (hsp 24) from chicken-embryo fibroblasts and co-migrated in one dimensional SDS gels with a major beta-crystallin was shown to be structurally similar to the chicken small heat shock protein but was distinct from the beta-crystallins.

Purification and properties of human platelet P235. A high molecular weight protein substrate of endogenous calcium-activated protease(s).

Two major high molecular weight proteins of human platelets are highly susceptible to proteolytic degradation by endogenous calcium-activated protease activities. Of the two proteins, one has been identified as filamin (Mr = 250,000 subunit); the second, a Mr = 235,000 subunit protein contributing 3-8% of the total platelet proteins, has not been previously characterized. We have now purified this protein, designated P235, to apparent homogeneity (greater than 95%). P235 was extracted by a Triton X-100 and EDTA containing buffer at pH 9.0 and purified by a series of DEAE-cellulose, phosphocellulose, and gel filtration chromatographies. Purified P235 is a dimer of Mr = 235,000 subunit. Its Stokes radius (67 A) and frictional ratio of 1.3 suggest that P235 is approximately globular. Despite this similarity in subunit and molecular weight of P235 to filamin, spectrin, fibronectin, and myosin, its amino acid composition, immunological properties, and peptide map are distinctly different and showed no precursor-product relationship to these proteins. Calcium-activated protease(s) in crude platelet extract rapidly degrade P235 into a Mr = 200,000 stable fragment. Upon prolonged storage at 4 degrees C, purified P235 partially degrades into a Mr = 220,000 and a Mr = 200,000 fragment. This degradation pattern suggests that P235 contains a large Mr = 200,000 protease-resistant domain. The availability of pure P235 will be useful in elucidating the functional role of this platelet protein, as well as the role of calcium-activated proteases in platelet function.