Molecular attenuation of vaccinia virus: mutant generation and animal characterization.

These studies demonstrated that the inbred BALB/c mouse strain can be optimized for the assessment of vaccinia virus virulence, growth, and spread from the site of inoculation and immune protection from a lethal vaccinia virus challenge. The studies established that manipulation of the vaccinia virus genome generated mutants exhibiting a wide range of attenuated phenotypes. The nine NYCBH vaccinia virus mutants had intracranial 50% lethal doses that ranged from 2 to greater than 7 log10 units. The decreased neurovirulence was due to decreased replication in brain tissue. Three mutants had a decreased ability to disseminate to the lungs, brains, livers, and spleens of mice after intranasal infection. One mutant had a decreased transmission from mice infected by tail scarification to naive cage mates. Although the mutants, with one exception, grew to wild-type titers in cell culture, they showed a growth potential on the scarified skin of mice that was dramatically different from that of the wild-type virus. Consequently, all of the mutants had significantly compromised immunogenicities at low virus immunization doses compared with that of the wild-type virus. Conversely, at high immunization doses most mutants could induce an immune response similar to that of the wild-type virus. Three Wyeth vaccine strain mutants were also studied. Whereas the thymidine kinase, ribonucleotide reductase, and hemagglutinin mutants had a reduced virulence (50% lethal dose), only the thymidine kinase mutant retained its immunogenicity.     

Differences in relative growth rate in 11 grasses correlate with differences in chemical composition as determined by pyrolysis mass spectrometry

Summary Eleven grass species varying in potential relative growth rate (RGR) were investigated for differences in chemical composition by pyrolysis mass spectrometry. The spectral data revealed correlations between RGR and the relative composition of several biopolymers. Species with a low potential RGR contained relatively more cell wall material such as lignin, hemicellulose, cellulose, polysaccharide-bound ferulic acid and hydroxyproline-rich protein, whereas species with a high potential RGR showed relatively more cytoplasmic elements such as protein (other than those incorporated in cell walls) and sterols.     

Soil acidification as caused by the nitrogen uptake pattern of Scots pine (Pinus sylvestris)

Three-year-old Scots pine (Pinus sylvestris) trees were grown on a sandy forest soil in pots, with the objective to determine their NH₄/NO₃ uptake ratio and proton efflux. N was supplied in three NH₄-N/NO₃-N ratios, 3:1, 1:1 and 1:3, either as ¹⁵NH₄+¹⁴NO₃ or as ¹⁴NH₄+¹⁵NO₃. Total N and ¹⁵N acquisition of different plant parts were measured. Averaged over the whole tree, the NH₄/NO₃ uptake ratios throughout the growing season were found to be 4.2, 2.5, and 1.5 for the three application ratios, respectively. The excess cation-over-anion uptake value (C_a-A_a) appeared to be linearly related to the natural logarithm of the NH₄/NO₃ uptake ratio. Further, this uptake ratio was related to the NH₄/NO₃ ratio of the soil solution. From these relationship it was estimated that Scots pine exhibits an acidifying uptake pattern as long as the contribution of nitrate to the N nutrition is lower than 70%. Under field circumstances root uptake may cause soil acidification in the topsoil, containing the largest part of the root system, and soil alkalization in deeper soil layers.     

Flow cytometric multiparameter analysis of proliferating cell nuclear antigen/cyclin and Ki-67 antigen: a new view of the cell cycle

Flow cytometric multiparameter analysis of two proliferation-associated nuclear antigens (proliferating cell nuclear antigen (PCNA)/cyclin and Ki-67) was performed on seven human hematopoietic cell lines. PCNA/cyclin, an S phase-related antigen, was detected using an autoantibody and a fluorescein isothiocyanate-labeled anti-human antibody. The Ki-67 antigen, which in cycling cells is expressed with increasing levels during the S phase with a maximum in the M phase, was detected using a monoclonal antibody and a phycoerythrin-conjugated anti-mouse antibody. In some experiments the PCNA/Ki-67 staining was combined with a DNA stain, 7-amino actinomycin D, and simultaneous detection of the three stains was performed by a single laser flow cytometer. Using this technique four distinct cell populations, representing G1, S, G2, and M, respectively, could be demonstrated in cycling cells on the basis of their PCNA/cyclin and Ki-67 levels. The cell cycle phase specificity could be verified using metaphase (vinblastine, colcemide) and G2 phase (mitoxantrone) blocking agents, as well as by stainings with a mitosis-specific antibody (MPM-2). Also, G0 cells could be discriminated from G1 cells in analysis of a mixture of resting peripheral mononuclear blood cells and a proliferating cell line. This technique can be valuable in detailed cell cycle analysis, since all cell cycle phases can be visualized and calculated using a simple double staining procedure.     

The activity of one soluble component of the cell-free NADPH:O2 oxidoreductase of human neutrophils depends on guanosine 5'-O-(3-thio)triphosphate

Neutrophil NADPH:O2 oxidoreductase activity, essential in the killing of bacteria by neutrophils, can be elicited in a cell-free system that requires plasma membranes, cytosol and sodium dodecyl sulfate. In addition, GTP or its nonhydrolyzable analog guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) enhances NADPH oxidase activity. We investigated the mechanism of this effect of GTP gamma S in the cell-free system. Cytosol from human neutrophils was separated in three different soluble oxidase components (SOC I, SOC II, and SOC III). Previously we (Bolscher, B. G. J. M., Van Zwieten, R., Kramer, I. J. M., Weening, R. S., Verhoeven, A. J., and Roos, D. (1989) J. Clin. Invest. 83, 757-763) reported that the cytosol contains two components which act synergistically. We now report that one component (previously labeled SOC II) contains two different components that can be separated by ion exchange chromatography. Immunoblotting with antiserum B-1 (Volpp, B. D., Nauseef, W. M., and Clark, R. A. (1988) Science 242, 1295-1297), directed against a cytosolic complex capable of activating latent membranes in the cell-free system, showed a 47-kDa protein in SOC II and a 67-kDa protein in SOC III. SOC II also contains the 47-kDa phosphoprotein, which indicates that this phosphoprotein and the protein recognized by the antiserum are identical. Low rates of NADPH-dependent O2 consumption can be elicited by SOC II and SOC III in the absence of SOC I. This activity is independent of GTP gamma S. Addition of SOC I increases this activity 3-4-fold, only when GTP gamma S is present. Plasma membranes, incubated with SOC I plus GTP gamma S and re-isolated, showed a similar 3-4-fold enhanced O2 consumption with SOC II and SOC III. The GTP gamma S effect is exerted primarily at the level of the plasma membrane. The concentration of GTP gamma S that causes a half-maximal stimulation was 0.4 mu M. It is concluded that SOC I is a functional component of the NADPH oxidase.     

Gangliosides in various brain areas of three inbred strains of mice

The ganglioside patterns of cerebellum, cortex, pons-medulla, hypothalamus, hippocampus and caudate nucleus of three inbred strains of mice (C57BL/6J, DBA/2J and BALB/cJ) have been analysed. All brain areas contained both the simple and complex species of gangliosides. GD1a was the major ganglioside in cortex, hippocampus and caudate nucleus whereas GT1b was the major species in cerebellum, hypothalamus and pons-medulla. In hippocampus, the percentages of GT1b and GD1a were quite similar. Pons and medulla exhibited the highest levels of GM1 (which approaches the value of GT1b) and the lowest values of GD1a. A ganglioside, termed here GT1L, was located between GD1b and GT1b. This ganglioside, which was present in highest amounts in cerebellum disappeared after alkali treatment. Highly significant differences were observed in the amounts and patterns of gangliosides among brain areas of the three strains. Highly significant differences (p<0.001) were also found in the ganglioside distribution of various brain areas among the strains, especially for tri-and tetrasialogangliosides between Balb and DBA. A significant difference of GM1 was observed in the cerebellum when comparing DBA with the two other strains. It is likely that the differences might be related to their relative abundances in certain cell types and for defining synaptic circuits in brain areas of some strains.     

Dehydration effects on imbibitional leakage from desiccation-sensitive seeds

Changes in electrolyte leakage and viability in response to dehydration stress were examined in two species of seeds that do not survive desiccation. Leakage from silver maple (Acer saccharinum L.) seeds increased markedly as seed moisture contents decreased from 45 to 35% (fresh weight basis) and germination decreased from 97 to 5%, coincidentally. Time course curves of imbibitional leakage from areca palm (Chrysalido-carpus lutescens [Bory] Wendl.) embryos showed an increase in both initial leakage and steady-state leakage rates in response to dehydration from an original moisture content of 84 to as low as 53%. Absorbance at 530 nanometers of extracts from triphenyl tetrazolium chloride stained embryos of areca palm was used as a measure of viability. Absorbance decreased significantly in response to dehydration as embryo moisture content decreased from 80 to 30%. Collectively, the data suggest that membranes in the desiccation-sensitive seed tissues studied are damaged by dehydration below a critical moisture content, 40% in silver maple seed and 55% in areca palm embryos, and that the membrane damage contributes to loss of viability.     

Isolation of human pituitary prolactin

A process developed earlier for the extraction of human follitropin, lutropin, thyrotropin and growth hormone from homogenized frozen pituitaries provided a residue utilized for the isolation of prolactin. The isolation procedure involved extraction at pH 9.8, molecular sieve chromatography on Sepharose CL-6B, hydrophobic interaction chromatography on phenyl-Sepharose CL-4B, molecular sieve chromatography on Sephadex G-100 Superfine, and ion-exchange chromatography on DEAE-Sepharose CL-6B using a convex gradient. The progressive purification was guided by radioimmunoassays. The final product was obtained in yields of 31 microgram/gland, and was equipotent with a pituitary preparation (VLS-3) supplied by the National Pituitary Agency (NIH, Bethesda, U.S.A.). Contamination hormones negligible (less than 0.05%). No heterogeneity of the isolated prolactin was observed by sedimentation-equilibrium analysis in the ultracentrifuge, by SDS electrophoresis in polyacrylamide gel or by molecular sieve chromatography in 6 M guanidine hydrochloride. These different techniques gave values in the range of 21 000-23 000 for the molecular weight of prolactin. In free zone electrophoresis, and also in polyacrylamide gel electrophoresis the prolactin preparation was, however, heterogeneous and resolved at alkaline pH into three distinct components. The former technique permitted isolation and assay of the components, indicating that they were all fully active.     

Characterization and quantification of the peroxidase in human monocytes.

1. The lifetime of thiamine pyrophosphate-Sepharose 2B affinity matrices synthesized according to Matsuura et al. (Matsuura, A., Iwashina, A. and Nose, Y. (1973) Biochem. Biophys. Res. Commun. 51, 241-246) has been improved. The matrix interacts with bacterial pyruvate dehydrogenase complexes. 2. The synthesis of a stable thiochrome-Sepharose 2B matrix is described. 3. Both matrices bind the pyruvate dehydrogenase complex of Escherichia coli in a 50 mM phosphate buffer, pH 7.0. Elution is possibly by an increase in ionic strength but not by the cofactor or metal-cofactor complexes. 4. The presence of Mg2+, reduces the capacity of the affinity matrices but leads to higher specificity for the multienzyme complex. 5. The pyruvate dehydrogenase complex of E. coli has been successfully purified by combining a classical purification step with these affinity chromatography systems. The method is less suitable for large scale operation.