Inhibition of the formation of lipid-linked intermediates in normal and transformed cells by a purified tunicamycin homologue

Summary The deffects of a purified homologue of tunicamycin (B₂-tunicamycin) on the biosynthesis of lipid-linked intermediates participating in protein glycosylation in normal embryonic fibroblasts, 3T3 and virally transformed (simian virus 40 and polyoma virus) mouse fibroblasts grown in culture were investigated. Long incubations (20 h) with the antibiotic caused a higher degree of inhibition of sugar incorporation into glycoproteins in transformed cells. However, the formation of lipid-linked intermediates was inhibited to a similar level in both cell types. When time dependent inhibition experiments were carried out using transformed cells, an earlier and stronger inhibition of the formation of lipid-oligosaccharides occurred (70% inhibition at 30 min). In 3T3 cells, prolonged incubation (6–8 h) was necessary in order to reach a similar degree of inhibition. Formation of lipid-sugar was also inhibited to a greater extent by B₂-tunicamycin in transformed cells. This inhibition was not clearly time dependent. Analysis of the newly synthesized glycolipids in 3T3 and in transformed cells after B₂-tunicamycin treatment have shown reduction in dolichyl-P-P-sugars as well as in other glycolipids. Dimethylsulfoxide (10%) and linoleic acid (0.5 mg/ml) markedly increased the level of tunicamycin activity in 3T3 cells while phosphatidylcholine (2 mg/ml) partially reversed it. The stronger and faster inhibition of the formation of lipid intermediates of the dolichyl-phosphate cycle caused by B₂-tunicamycin in transformed cells, described here for the first time, may therefore be due to differences in penetration of the antibiotic into these cells.Abbreviations DMEM Dulbecco's modified Eagle's medium - DMSO dimethylsulfoxide - MF mouse fibroblasts from Balb/c mouse embryos - 3T3 Balb/3T3 mouse fibroblastic line - SV40 Simian virus 40 - PY polyoma virus - TLC thin layer chromatography     

Effect of melphalan in vitro on induction of murine suppressor T cells by ConA

Summary The effect of treatment with melphalan in vitro on the activity of spleen cells from BALB/c mice was investigated. Incubation of spleen cells with 1.5–5 g melphalan/1×10⁷ inhibited subsequent mitogenic stimulation by ConA or PHA and the allogeneic response of BALB/c spleen cells against C57B1 target spleen cells. Incubation of spleen cells with ConA led to induction of suppressor T cells which when added to fresh cultures inhibited the allogeneic response. Preincubation of spleen cells with melphalan even at low concentrations (0.15–0.5 g 1×10⁷ cells) which do not directly affect mitogenic stimulation or allogeneic response partially inhibited the generation of suppressor T cells by ConA. Treatment with melphalan had no effect on already induced suppressor T cells as shown by incubation of spleen cells with melphalan (0.15–5 g/1×10⁷ cells) after incubation with ConA. Addition of cells treated with melphalan alone (without ConA) to fresh cultures led to an increase in the allogeneic response.     

Ordered water structure in an A-DNA octamer at 1.7 A resolution

The crystal structure of the deoxyoctamer d(G-G-Br U-A-BrU-A-C-C) was refined to a resolution of 1.7 A using combined diffractometer and synchrotron data. The analysis was carried out independently in two laboratories using different procedures. Although the final results are identical the comparison of the two approaches highlights potential problems in the refinement of oligonucleotides when only limited data are available. As part of the analysis the positions of 84 solvent molecules in the asymmetric unit were established. The DNA molecule is highly solvated, particularly the phosphate-sugar back-bone and the functional groups of the bases. The major groove contains, in the central BrU-A-BrU-A region, a ribbon of water molecules forming closed pentagons with shared edges. These water molecules are linked to the base O and N atoms and to the solvent chains connecting the O-1 phosphate oxygen atoms on each strand. The minor groove is also extensively hydrated with a continuous network in the central region and other networks at each end. The pattern of hydration is briefly compared with that observed in the structure of a B-dodecamer.     

Purification of human platelet calcium-activated protease. Effect on platelet and endothelial function

Calcium-activated protease (CAP) was purified from the cytosol fraction of homogenized human platelet concentrates using a combination of gel filtration chromatography and affinity chromatography on antipain aminohexyl-Sepharose and activated thiol-Sepharose 4B. Purified CAP is composed of two different polypeptides of Mr = 80,000 and 27,000. Half-maximal protease activity was observed at 0.52 mM Ca2+, and all activity was inhibited by antipain, leupeptin, and N-ethylmaleimide. Activated CAP showed a time-dependent inactivation in the presence of 1 mM Ca2+ with only 5% of the control protease activity remaining after a 1-h exposure to calcium. Preincubation of washed platelets with varying amounts of CAP (0.2-0.4 units) significantly interfered with thrombin-induced platelet aggregation. In addition, ristocetin-induced platelet agglutination in the presence of von Willebrand factor was completely inhibited by 0.4 units of CAP. Concomitant with these protease-induced changes in platelet function, a decrease was observed in a major glycoprotein band of Mr = 150,000 present in platelet membranes and presumed to be glycoprotein Ib. In addition to these effects on platelets, CAP inhibited thrombin-induced production of prostacyclin by cultured human endothelial cells in a dose-dependent manner when the cells were pretreated with CAP. Thus platelet CAP can modulate membrane functions in both platelets and endothelial cells and may thus contribute to the regulation of hemostasis.     

13C NMR Analysis of some simple tetrahydroisoquinolines

¹³C NMR resonances of 15 simple tetrahydroisoquinolines have been assigned on the basis of chemical shift theory, ¹³C-¹H coupling constants     

Chloroplast and cytoplasmic low-molecular-weight ribonucleic acid components of the leaf of Vicia faba L

1. A method for the extraction of plant nucleic acids and their separation on methylated-serum-albumin-kieselguhr columns is described. It is demonstrated that the characteristics of the elution profiles of material from the same source are consistently reproducible. 2. Major dissimilarities were found in the elution profiles of nucleic acids from root and from leaves of Vicia faba L. These dissimilarities were confirmed by polyacrylamide-gel electrophoresis. 3. Four distinct types of low-molecular-weight RNA were demonstrated to be present in leaves, clearly distinguished by their behaviour when chromatographed on methylated-serum-albumin-kieselguhr columns. (a) Both cytoplasmic and chloroplast ribosomes contained a low-molecular-weight RNA, and these components were distinct from each other. (b) The chloroplast possessed a unique ;soluble' RNA (i.e. RNA that is not precipitated by centrifugal forces that sediment ribosomes) which was not present in the rest of the cell. (c) A soluble component, probably transfer RNA, was found in both the chloroplasts and in the cytoplasm. 4. The components distinguishable by methylated-serum-albumin-kieselguhr column chromatography could not be distinguished by sucrose-density-gradient centrifugation.     

Factors influencing plasma zinc levels in low-income pregnant women

Plasma zinc (Zn) concentrations were measured in 4376 indigent women (86% African-American), at a mean (±SD) gestational age of 15 (±7.8) wk to determine the relationship between various maternal characteristics and plasma Zn levels during pregnancy. Mean plasma Zn levels were lower in African-American women than in Caucasian women, in multiparous women than in primiparous women, and in women with body weight >69.9 kg than in those with body weight ≤69.9 kg (p≤0.001 for each comparison). There were no significant differences related to maternal age, marital status, education, or smoking habit. Multiple regression analysis, including maternal prepregnancy weight, race, age, parity, smoking habit, education, and marital status indicated that race, parity, and pregnancy weight were significantly associated with maternal plasma Zn levels, adjusted for gestational age. Maternal race was the best predictor of plasma Zn concentrations among the population of pregnant women studied A significant proportion of variance in maternal plasma Zn levels remained unexplained after taking into account various maternal characteristics. The reasons for lower plasma Zn levels in African-American women, compared to Caucasian women, during pregnancy are unknown.     

Reproductive responses of Iphiseius degenerans and Neoseiulus teke (Acari: Phytoseiidae) to changes in the density of the cassava green mite, Mononychellus tanajoa (Acari: Tetranychidae)

We assessed the reproductive responses of adult female Iphiseius degenerans and Neoseiulus teke to increasing density of three stages of their prey, Mononychellus tanajoa, on cassava leaf discs under laboratory conditions. The oviposition rates increased with number of prey consumed per predator per day with a maximum of approximately two eggs per day for I. degenerans and four eggs per day for N. teke. The oviposition rate of N. teke was higher when consuming eggs than other prey stages. Neoseiulus teke was more efficient than I. degenerans in converting consumed prey into egg production. The data were adequately described by simple mathematical models.     

Purified moniliformin does not affect the force or rate of contraction of isolated guinea pig atria

Aspergillus flavus Link ex Fries and A. parasiticus Speare can invade peanut kernels and under certain environmental conditions produce unacceptable levels of the mycotoxin aflatoxin. A concerted effort is underway to reduce aflatoxin contamination in peanut and peanut products. A potentially effective method of control in peanut is the discovery and use of genes for resistance to either fungal invasion or aflatoxin formation. The objective of the present experimental study was to develop an effective and efficient procedure for screening individual plants or pods of single plants for resistance to invasion by the aflatoxigenic fungi and subsequent aflatoxin production. Methods of obtaining adequate drought-stress and fungal infection were developed through this series of experiments. By completely isolating the pods from the root zone and imposing drought-stress only on pegs and pods, high levels of fungal infection were observed. High amounts of preharvest aflatoxin accumulation were also produced by completely isolating the pods from the root zone. Mid-bloom inoculation with A. parasiticus-contaminated cracked corn and drought-stress periods of 40 to 60 days were the most effective procedures. This technique was used to assess peanut genotypes previously identified as being partially resistant to A. parasiticus infection or aflatoxin contamination, and segregating populations from four crosses. Variability in aflatoxin contamination was found among the 11 genotypes evaluated, however, none were significantly lower than the standard cultivars. Broad-sense heritability of four crosses was estimated through evaluation of seed from individual plants in the F₂ generation. The heritability estimates of crosses GFA-2 × NC-V11 and Tifton-8 × NC-V11 were 0.46 and 0.29, respectively, but mean aflatoxin contamination levels were high (73,295 and 27,305 ppb). This greenhouse screening method could be an effective tool when genes for superior aflatoxin resistance are identified.Cooperative investigation of the USDA-ARS and the University of Georgia, College of Agriculture.