Involvement of the 2'-5' A pathway in the augmentation of natural killer activity

Pretreatment of human large granular lymphocytes (LGL) or unseparated peripheral blood mononuclear cells with interferon (IFN) resulted in a significant augmentation of natural killer (NK) activity. This increase was paralleled by an increase in the 2'-5'A synthetase activity. In order to investigate the possibility that IFN might be inducing augmentation of NK cells via the 2'-5'A pathway, we tested the effects of nonphosphorylated core material [(A2'p)2A] and of the triphosphorylated form of the 2'-5'A [ppp(A2'p)2A]. The core material had no detectable effect on NK activity. In contrast, when experiments were performed with the triphosphorylated form of 2'-5'A, NK activity was stimulated. In order to achieve activation, permeabilization of LGL with calcium chloride was necessary and, under these conditions, a dose-dependent augmentation of NK activity was seen. However, the calcium treatment had considerable toxic effects on basal levels of NK activity. Collectively, these results suggest that IFN may be inducing augmentation of NK activity via the 2'-5'A pathway. Further studies will be necessary to determine the effects of IFN and/or 2'-5'A on subsequent activation steps in the process leading to cytotoxicity by NK cells.     

Sulfur metabolism in Beggiatoa alba

The metabolism of sulfide, sulfur, and acetate by Beggiatoa alba was investigated under oxic and anoxic conditions. B. alba oxidized acetate to carbon dioxide with the stoichiometric reduction of oxygen to water. In vivo acetate oxidation was suppressed by sulfide and by several classic respiratory inhibitors, including dibromothymoquinone, an inhibitor specific for ubiquinones. B. alba also carried out an oxygen-dependent conversion of sulfide to sulfur, a reaction that was inhibited by several electron transport inhibitors but not by dibromothymoquinone, indicating that the electrons released from sulfide oxidation were shuttled to oxygen without the involvement of ubiquinones. Intracellular sulfur stored by B. alba was not oxidized to sulfate or converted to an external soluble form under aerobic conditions. On the other hand, sulfur stored by filaments of Thiothrix nivea was oxidized to extracellular soluble oxidation products, including sulfate. Sulfur stored by filaments of B. alba, however, was reduced to sulfide under short-term anoxic conditions. This anaerobic reduction of sulfur was linked to the endogenous oxidation of stored carbon and to hydrogen oxidation.     

Interaction of small dermatan sulfate proteoglycan from fibroblasts with fibronectin

Immunogold labeling was used to localize the core protein of small dermatan sulfate proteoglycan (DS-PG) on the surface of cultured human fibroblasts. At 4 degrees C, DS-PG core protein was uniformly distributed over the cell surface. At 37 degrees C, gold particles either became rearranged in form of clusters or remained associated with fibrils. Double-label immunocytochemistry indicated the co-distribution of DS-PG core protein and fibronectin in the fibrils. In an enzyme-linked immunosorbent assay, binding of DS-PG from fibroblast secretions and of its core protein to fibronectin occurred at pH 7.4 and at physiological ionic strength. Larger amounts of core protein than of intact proteoglycan could be bound. Fibronectin peptides containing either the heparin-binding domain near the COOH-terminal end or the heparin-binding NH2 terminus were the only fragments interacting with DS-PG and core protein. Competition and replacement experiments with heparin and dermatan sulfate suggested the existence of adjacent binding sites for heparin and DS-PG core protein. It is hypothesized that heparan sulfate proteoglycans and DS-PG may competitively interact with fibronectin.     

GC-MS identification of GA20-13-O-glucoside formed from GA20 in normal plants and dwarf-1 mutants ofZea mays L.

After feeding GA₂₀ to excised seedlings ofZea mays L. normals (N) and dwarf-1 mutants (d1), GA₂₀-13-O-glucoside (9) was identified by HPLC and by GC-MS of its permethylated derivative. The glucosylation rate of GA₂₀ was found to be higher in the dwarf-1 mutant (26%) than in the normal plant (3.6%). This article includes a GC-MS study in which diagnostic fragments from the spectra of permethylated synthetic GA glucosides have been selected that proved to be useful for the identification of permethylated GA glucosides.     

Internal motions in yeast phenylalanine transfer RNA from 13C NMR relaxation rates of modified base methyl groups: a model-free approach

Internal motions at specific locations through yeast phenylalanine tRNA were measured by using nucleic acid biosynthetically enriched in 13C at modified base methyl groups. Carbon NMR spectra of isotopically enriched tRNA(Phe) reveal 12 individual peaks for 13 of the 14 methyl groups known to be present. The two methyls of N2,N2-dimethylguanosine (m22G-26) have indistinguishable resonances, whereas the fourteenth methyl bound to ring carbon-11 of the hypermodified nucleoside 3' adjacent to the anticodon, wyosine (Y-37), does not come from the [methyl-13C]methionine substrate. Assignments to individual nucleosides within the tRNA were made on the basis of chemical shifts of the mononucleosides [Agris, P. F., Kovacs, S. A. H., Smith, C., Kopper, R. A., & Schmidt, P. G. (1983) Biochemistry 22, 1402-1408; Smith, C., Schmidt, P. G., Petsch, J., & Agris, P. F. (1985) Biochemistry 24, 1434-1440] and correlation of 13C resonances with proton NMR chemical shifts via two-dimensional heteronuclear proton-carbon correlation spectroscopy [Agris, P. F., Sierzputowska-Gracz, H., & Smith, C. (1986) Biochemistry 25, 5126-5131]. Values of 13C longitudinal relaxation (T1) and the nuclear Overhauser enhancements (NOE) were determined at 22.5, 75.5, and 118 MHz for tRNA(Phe) in a physiological buffer solution with 10 mM MgCl2, at 22 degrees C. These data were used to extract two physical parameters that define the system with regard to fast internal motion: the generalized order parameters (S2) and effective correlation times (tau e) for internal motion of the C-H internuclear vectors.(ABSTRACT TRUNCATED AT 250 WORDS)     

The genotypic response of mouse embryos to multiple freezing variables

Four- and eight-cell embryos from 3 mouse genotypes were cryopreserved to study the relationship of genetics and freezing variables on post-thaw survival. Embryos from outbred N:NIH(S), N:NIH(S)-B and inbred (C57BL/6N) mice were exposed to 1 of 2 cryoprotectants (glycerol [GLYC] versus dimethyl sulfoxide [DMSO]) and stored in 1 of 2 containers (ampules [AMP] versus straws [STR]). Containerized embryos were cooled at a rate of 0.5 degrees C/min to a minimum of -35 degrees C, transferred into liquid nitrogen, and later thawed and cultured in vitro. Genotypic differences (p less than 0.05) were noted for 4 interrelated embryo characteristics including post-thaw survival (PTS), embryo degeneration rate (EDR), and quality grade (QG) and viability grade (VG) ratings. The PTS for outbred embryos was greater (p less than 0.05) in GLYC than DMSO, whereas inbred C57BL/6N embryos survived similarly (p greater than 0.05) in either cryoprotectant. Compared to DMSO counterparts, embryos from GLYC-treated outbred groups had improved QG and VG ratings and reduced EDR (p less than 0.05), but comparable results were observed between GLYC- AND DMSO-treated embryos in the C57BL/6N group. Between genotypes, type of container affected PTS and EDR (p less than 0.05) but not QG or VG ratings (p greater than 0.05). Within genotypes, PTS generally ranged 15 to 20% higher (p less than 0.01) in AMP than STR groups. Increased PTS was noted in outbred mouse x GLYC x AMP groups; however, based on the degrees of difference, the inbred C57BL/6N strain appeared less affected by this 3-way interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Continuous production of L-phenylalanine by transamination

L-Phenylalanine was produced continuously from L-as-partate and phenylpyruvate by transaminase from a newly screened Pseudomonas putida strain. The process was carried out with an isolated enzyme in homogeneous phase in an enzyme membrane reactor and with immobilized whole cells in a stirred tank reactor, respectively. Due to the difference in transport resistance, the productivity of the free enzyme in homogeneous phase (72 mmol/L h) was about 3 times higher than the productivity achieved using immobilized cells. However, a better stability of the biocatalyst was observed with immobilized cells.     

The maize autonomous element Activator (Ac) shows a minimal germinal excision frequency of 0.2%–0.5% in transgenic Arabidopsis thaliana plants

Summary The autonomous mobile element Activator from Zea mays was introduced into Arabidopsis thaliana via Agrobacterium-mediated gene transfer. The use of a chimaeric construct, where the Ac element is located in the leader of the neomycin phosphotransferase (NPT II) gene, enabled the excision of Ac to be monitored by assaying for the reconstitution of NPT II gene activity. Using this approach, the transpositional activity of AC was initially studied in primary transformants. About 50% of the regenerating Ac transformants showed evidence for excision of the element. Reintegration of Ac was confirmed by Southern blot analysis. Transposition events are transmitted to the F1 generation with a minimal frequency of 0.3%. In a few exceptional cases they are detected in a high proportion of the F1 generation. Seedlings from the F2 and F3 generations were assayed for the rate of germinal excisions by scoring for kanamycin resistance. The minimal frequency of germinal excision events amounts to 0.2%–0.5% and hence allows the use of the Ac element for gene tagging purposes in A. thaliana.     

Studies of the oxysterol inhibition of tumor cell growth

The oxysterols 3 beta-hydroxy-5 alpha-cholest-8-en-11-one, 3 beta-hydroxy-5 alpha-cholest-8-en-7-one, 3 beta-hydroxy-5 alpha-cholest-8(14)-en-7-one, 3 beta-hydroxy-4,4'-dimethylcholest-5-ene-7 one, 4,4'-dimethylcholest-5-ene-3 beta, 7 alpha-diol, 4,4'-dimethylcholest-5-ene-3 beta, 7 beta-diol, lanost-8-ene-3 beta, 25-diol, 25-hydroxylanost-8-en-3-one, 9 alpha, 11 alpha-epoxy-5 alpha-cholest-7-en-3 beta-ol, 3 beta-hydroxycholest-5 alpha-en-22-one, and 3 beta-hydroxycholest-5-en-22-one oxime were evaluated with respect to their ability to inhibit cell growth. All of the sterols were found to possess cytotoxicity when incubated with hepatoma (HTC) and lymphoma (RDM-4) cells in culture at 10-30 microM concentrations.