Characterization of specific high affinity receptors for human tumor necrosis factor on mouse fibroblasts

Mouse L-929 fibroblasts, an established line of cells, are very sensitive to lysis by human lymphotoxin (hTNF-beta). Specific binding of a highly purified preparation of hTNF-beta to these cells was examined. Recombinant DNA-derived hTNF-beta was radiolabeled with [3H]propionyl succinimidate at the lysine residues of the molecule to a specific activity of 200 microCi/nmol of protein. [3H]hTNF-beta was purified by high performance gel permeation chromatography and the major fraction was found to be monomeric by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The labeled hTNF-beta was fully active in causing lysis of L-929 fibroblasts and bound specifically to high affinity binding sites on these cells. Scatchard analysis of the binding data revealed the presence of a single class of high affinity receptors with an apparent Kd of 6.7 X 10(-11) M and a capacity of 3200 binding sites/cell. Unlabeled recombinant DNA-derived hTNF-beta was found to be approximately 5-fold more effective competitive inhibitor of binding than the natural hTNF-beta. The binding of hTNF-beta to these mouse fibroblasts was also correlated with the ultimate cell lysis. Neutralizing polyclonal antibodies to hTNF-beta efficiently inhibited the binding of [3H]hTNF-beta to the cells. We conclude that the specific high affinity binding site is the receptor for hTNF-beta and may be involved in lysis of cells.     

An osmotic mechanism for exocytosis from dissociated chromaffin cells

Dissociated chromaffin cells from bovine adrenal medulla were stimulated to secrete epinephrine and dopamine beta-hydroxylase with a variety of secretagogues in a study designed to test the hypothesis that the chemiosmotic lysis reaction of isolated chromaffin granules might in some way be related to the mechanism of release during exocytosis. Increasing the osmotic strength of the incubation medium with either NaCl or sucrose led to suppression of secretion of epinephrine from the cells regardless of whether secretion was induced with veratridine or acetylcholine. Suppression of secretion was approximately exponential with respect to osmotic strength. Epinephrine secretion occurred only if the medium contained a permeant anion such as chloride, and secretion induced by veratridine was suppressed when Na isethionate replaced NaCl in the medium. In an extensive study with different monovalent anions veratridine supported epinephrine secretion according to the following activity series: Br-, I-, NO3- greater than methylsulfate, SCN- greater than Cl greater than acetate much greater than isethionate. A similar series, except for the potency of NO3-, was observed with A23187 as agonist. In general, the anion series for granule lysis was analogous. However, there was a poor quantitative correlation between the anion dependence of chemiosmotic granule lysis and the anion dependence of cell secretion. Anion transport inhibitors such as probenecid and pyridoxal phosphate also inhibited secretion while the stilbene disulfonates were inactive. The ineffectiveness of the stilbene disulfonates further distinguished chemiosmotic granule lysis from cell secretion. Secretion of catecholamines, induced by veratridine or nicotine, a cholinergic agonist, was suppressed when NaCl in the medium was replaced by isosmotic sucrose and unexpectedly low levels of dopamine beta-hydroxylase were observed in some cases. In sum, these properties of secreting chromaffin cells resembled some properties of isolated chromaffin granules incubated in ATP and Cl-, but were different in a number of instances. We, therefore, have interpreted our data to indicate that while some mechanistic relationships may indeed exist between the release event in exocytosis from chromaffin cells and the chemiosmotic lysis reaction characteristic of isolated chromaffin granules, an understanding of the energetics of exocytosis awaits the discovery of reasons for the quantitative differences between the two systems.     

Chromosome damage in rat pulmonary alveolar macrophages following ozone inhalation

To determine whether ozone is clastogenic at environmentally relevant exposure levels, rats were exposed for 6 h to 0.0, 0.12, 0.27, or 0.80 ppm ozone. The alveolar macrophages were isolated from animals sacrificed 28 h after the end of the exposure. The mitotic index and frequency of chromosome aberrations were determined. No change in the mitotic index was detected following 0.12 ppm ozone exposure. A significant decrease in mitotic index was observed after exposure to 0.27 ppm ozone; a significant (4-fold) increase in the frequency of dividing macrophages was detected following exposure to 0.8 ppm ozone. Only chromatid-type aberrations were observed. There was a significant increase in the frequency of cells with chromatid gaps and in the frequency of cells with chromatid deletions. Animals exposed to 0.27 ppm ozone had the highest proportion of cells with chromatid deletions (0.172) relative to background level (0.028). No exchanges or chromosome-type aberrations were detected in any of the animals. These data suggest that ozone, at relatively low levels, is clastogenic in macrophages from exposed rats.     

THE COMPOSITION AND PHYLOGENETIC SIGNIFICANCE OF THE MOUGEOTIA (CHAROPHYCEAE) CELL WALL1

The two-layered, fibrillar cell wall of Mougeotia C. Agardh sp. consisted of 63.6% non-cellulosic carbohydrates and 13.4% cellulose. The orientation of cellulose microfibrils in the native cell wall agrees with the multinet growth hypothesis, which has been employed to explain the shift in microfibril orientation from transverse (inner wall) toward axial (outer wall). Monosaccharide analysis of isolated cell walls revealed the presence of ten sugars with glucose, xylose and galactose most abundant. Methylation analysis of the acid-modified, 1 N NaOH insoluble residue fraction showed that it was composed almost exclusively of 4-linked glucose, confirming the presence of cellulose. The major hemicellulosic carbohydrate was semi-purified by DEAE Sephacel (Cl^?) anion-exchange chromatography of the hot 1 N NaOH soluble fraction. This hemicellulose was a xylan consisting of a 4-xylosyl backbone and 2,4-xylosyl branch points. The major hot water soluble neutral polysaccharide was identified as a 3-linked galactan. Mougeotia cell wall composition is similar to that of (Charophyceae) and has homologies with vascular plant cell walls. Our observations support transtructural evidence which suggests that members of the Charophyceae represent the phylogenetic line that gave rise to vascular plants. Therefore, the primary cell walls of vascular plants many have evolved directly from structures typical of the filamentous green algal cell walls found in the Charophyceae.     

An Ustilago maydis Mutant Partially Blocked in P45014DM Activity Is Hypersensitive to Azole Fungicides

An Ustilago maydis ergosterol biosynthesis mutant (A14) which is partially blocked in sterol 14alpha-demethylase (P45014DM) activity is described. This mutant accumulated the abnormal 14alpha-methyl sterols, eburicol, 14alpha-methylfecosterol, and obtusifoliol, along with significant amounts of ergosterol. Although the A14 mutant grew nearly as well as the wild type, it was impaired in cell extension growth, which indicated a dysfunction in apical cell wall synthesis. The mutant was also found to be hypersensitive to the azole fungicides penconazole and tebuconazole.     

The long-term effects of multiple doses of methamphetamine on neostriatal tryptophan hydroxylase, tyrosine hydroxylase, choline acetyltransferase and glutamate decarboxylase activities.

The long-term effects of methamphetamine (15 mg/kg, s.c.) on neostriatal enzyme activity were measured in rats treated every 6 hours for 24 hours and subsequently monitored for periods up to 30 days following administration of the drug. Tyrosine hydroxylase was maximally decreased after the first day of drug treatment but the depression persisted throughout a 30-day period. Within 15 hours after methamphetamine treatment was initiated, trytophan hydroxylase was dramatically depressed to 17% of control and was still maximally depressed 7.5 days after the last dose of the drug. In contrast, neostriatal choline acetyltransferase and glutamate decarboxylase activities were not altered acutely nor chronically by methamphetamine administration. It would appear that the toxic effects of methamphetamine reported previously cannot be attributed to a generalized destruction of the neostriatum but are demonstrable only in selected biogenic amine nerve terminals.     

Cation-stimulated Adenosine Triphosphatase Activity and Cation Transport in Corn Roots

ATPase activity of the plasma membrane fraction from primary roots of corn (Zea mays L. WF9 x M14) was activated by Mg(2+) and further stimulated by monovalent cations (K(+) > Rb(+) > Cs(+) > Na(+) > Li(+)). K(+)-stimulated activity required Mg(2+) and was substrate-specific. Maximum ATPase activity in the presence of Mg(2+) and K(+) was at pH 6.5 and 40 C. Calcium and lanthanum (<0.5 mm) were inhibitors of ATPase, but only in the presence of Mg(2+). Oligomycin was not an inhibitor of the plasma membrane ATPase, whereas N,N'-dicyclohexylcarbodiimide was. Activity showed a simple Michaelis-Menten saturation with increasing ATP.Mg. The major effect of K(+) in stimulating ATPase activity was on maximum velocity. The kinetic data of K(+) stimulation were complex, but similar to the kinetics of short term K(+) influx in corn roots. Both K(+)-ATPase and K(+) influx kinetics met all criteria for negative cooperativity. The results provided further support for the concept that cation transport in plants is energized by ATP, and mediated by a cation-ATPase on the plasma membrane.     

Hexazonium pararosaniline as a fixative for animal tissues

Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a “fixative” action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution.     

Influence of Age on the Fatty Acid Composition of Breast and Thigh Muscles of Male Turkeys

Large White male turkeys were sacrificed at 4-week intervals from 4 to 28 weeks of age to study the fatty acid distribution in lipid of breast and thigh muscles. A total of 70 turkeys were sampled for this experiment.

Fatty acid distribution varied with advancing maturity and between muscle types. The most abundant fatty acids in the tissues were those with carbon chain lengths of 15:0, 16:0, 18:0, 18:1, 18:2, 20:4 and 24:0. Thigh muscle contained significantly more linoleate (18:2) than did breast. Larger proportions of pentadecanoic (15:0), arachidonic (20:4) and lignoceric (24:0), however, appeared in breast. Indications of minor fatty acids appeared on the chromatograms, but their low concentrations made their estimation and identification difficult.