Poly(I):poly(C)-enhanced alveolar and peritoneal macrophage phagocytosis: quantification by a new method utilizing fluorescent beads

Phagocytosis is an important immune function to quantify. This immune response may be modulated by exposure to biological response modifiers or by exposure to pollutants. A new technique for quantifying nonspecific phagocytosis of alveolar and peritoneal macrophages in the same animal has been developed that utilizes fluorescent polystyrene beads. When incorporated into inhalation studies, this technique can be used to determine whether the toxic effect of an inhaled pollutant is local (effect on alveolar macrophages), systemic (effect on peritoneal macrophages), or both local and systemic. This method results in a determination of both the level of phagocytosis (the percentage of phagocytic macrophages) and the macrophage specific activity (the number of beads phagocytized per macrophage). This method also allows a determination of adherence by quantifying the number of particles in contact with, but not phagocytized by, the macrophage. Macrophage preparations were incubated with fluorescent beads for 2 hr and cyto-centrifuged onto a glass slide. Fluorescent beads present on the slide or cell-associated but not ingested by phagocytosis were removed by immersing the slide containing the macrophage preparation in methylene chloride for 15-30 sec. Fluorescent beads ingested by phagocytosis were then easily quantified with a fluorescence microscope. This technique was used to assess the baseline levels of phagocytosis for rat alveolar and peritoneal macrophages from the same animal and the kinetics and level of enhanced phagocytosis for alveolar and peritoneal macrophages after injection with the interferon inducer polyinosinate-polycytidylate (poly(I):poly(C)). The kinetics of enhanced alveolar and peritoneal macrophage phagocytosis by poly(I):poly(C) were similar; however, stimulated phagocytic levels of peritoneal macrophages never reached the phagocytic activity observed for the resident, highly phagocytic alveolar macrophages. This elevated phagocytic activity is most likely due to interferon stimulated by particulate matter in the large volume of air processed by the lungs and is important for host defense against a number of different inhaled microorganisms.     

Aminoglycoside suppression at UAG, UAA and UGA codons in Escherichia coli and human tissue culture cells

Summary We have compared the suppression of nonsense mutations by aminoglycoside antibiotics inEscherichia coli and in human 293 cells. Six nonsense alleles of the chloramphenicol acetyl transferase (cat) gene, in the vector pRSVcat, were suppressed by growth in G418 and paromomycin. Readthrough at UAG, UAA and UGA codons was monitored with enzyme assays for chloramphenicol acetyl transferase (CAT), in stably transformed bacteria and during transient expression from the same plasmid in human 293 tissue culture cells. We have found significant differences in the degree of suppression amongst three UAG codons and two UAA codons in different mRNA contexts. However, the pattern of these effects are not the same in the two organisms. Our data suggest that context effects of nonsense suppression may operate under different rules inE. coli and human cells.     

Interacting proteins identified by genetic interactions: a missense mutation in alpha-tubulin fails to complement alleles of the testis-specific beta-tubulin gene of Drosophila melanogaster.

In this paper we demonstrate that failure to complement between mutations at separate loci can be used to identify genes that encode interacting structural proteins. A mutation (nc33) identified because it failed to complement mutant alleles of the gene encoding the testis-specific beta 2-tubulin of Drosophila melanogaster (B2t) did not map to the B2t locus. We show that this second-site noncomplementing mutation is a missense mutation in alpha-tubulin that results in substitution of methionine in place of valine at amino acid 177. Because alpha- and beta-tubulin form a heterodimer, our results suggest that the genetic interaction, failure to complement, is based on the structural interaction between the protein products of the two genes. Although the nc33 mutation failed to complement a null allele of B2t (B2tn), a deletion of the alpha-tubulin gene to which nc33 mapped complemented B2tn. Thus, the failure to complement appears to require the presence of the altered alpha-tubulin encoded by the nc33 allele, which may act as a structural poison when incorporated into either the tubulin heterodimer or microtubules.     

Neutron fibre diffraction study of DNA hydration

Interactions with water are crucial to the conformation assumed by the DNA double helix. The location of water around the D conformation has been investigated in a neutron fibre diffraction study which shows that water is ordered in the minor groove of the DNA. The D conformation is important since its occurrence is limited to specific DNA base pair sequences which have been identified as functionally significant. This study is of particular interest because the D conformation has not been reported in single crystal studies of oligonucleotides.     

Molecular structure of tetanus neurotoxin as revealed by Fourier transform infrared and circular dichroic spectroscopy

Secondary structure contents of tetanus neurotoxin have been estimated at neutral and acidic pH using circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. An analysis of the far-ultraviolet CD spectra of the neurotoxin dissolved in 50 mM citrate-phosphate buffer (pH 7.0) revealed 20.0 +/- 2.1% alpha-helix, 50.5 +/- 2.1% beta-pleated sheets, no beta-turns, and 29.5% random coils, which is at considerable variance with results from an earlier detailed study of tetanus neurotoxin's secondary structures (J.P. Robinson, L.A. Holladay, J.H. Hash and D. Puett, J. Biol. Chem. 257 (1982) 407). However, the alpha-helix content estimated in this study is consistent with the earlier studies of Robinson et al. (J.P. Robinson, L.A. Holladay, J.B. Picklesimer and D. Puett, Mol. Cell. Biochem. 5 (1974) 147; J.P. Robinson, J.B. Picklesimer and D. Puett, J. Biol. Chem. 250 (1975) 7435) and with the study by Lazarovici et al. (P. Lazarovici, P. Yanai and E. Yavin, J. Biol. Chem. 262 (1986) 2645), although other secondary structural features do not agree with those of the previous studies. Secondary structure estimation from Fourier transform infrared spectra in both amide I and amide III frequency regions revealed 22-23% alpha-helix, 49-51% beta-pleated sheets and 27-28% random coils, indicating a good correlation with the secondary structure content estimated from CD analysis. Lowering of the pH of the neurotoxin to 5.5 or 4.0 did not result in any noticeable change in the overall secondary structures. However, there were significant pH-induced variations observed in the individual curve-fitted FT-IR bands in the amide III frequency region. For example, the 1302 cm-1 band (relative area, 4.2%) observed at pH 7.0 was shifted to 1297 cm-1 (relative area, 2.2%) at pH 5.5, and the relative area of the band at 1316-1317 cm-1 (alpha-helix) increased by approx. 40%. This study suggests that contrary to earlier reports, tetanus neurotoxin is a beta-pleated sheet dominated structure, and although lower pH does not change the overall contents of the secondary structures, significant conformational alterations are observed.     

Light-temperature interactions on the growth of Antarctic diatoms

Summary The combined effect of various temperatures and light intensities on the growth of seven species of antarctic diatoms in culture has been studied. With the exception of Chaetoceros deflandrei whose thermal tolerance is fairly good, these obligatory psychrophils cannot survive in temperatures above 6° to 9° C. Their mean growth rate is relatively low, between 0.24 div d^–1 for Corethron criophilum and 0.63 div d^–1 for C. deflandrei. Regardless of light intensity, growth rate increased with the temperature to reach a maximum between 3° and 5° C. The highest rates were obtained between 115 and 220 mol m^–2 s^–1 with 0.38 div d^–1 for C. criophilum, 0.56 div d^–1 for Synedra sp. and between 0.71 and 0.88 div d^–1 for the other 5 species. A reduction in light intensity from 220 to 46 mol m^–2 s^–1 slowed growth by nearly 50%. These results suggest that the combined effect of temperature and light is one of the factors involved in the limitation of antarctic phytoplankton growth. The low temperatures of the environment do not permit rapid growth, which, even under optimal light conditions remains low. In addition, in the euphotic layer, the overall light energy available for algae is considerably reduced due to turbulence, a factor which exacerbates the reduced growth rate.     

Auxin activity of phenylacetic acid in tissue culture

The ability of phenylacetic acid (PAA), a naturally occurring auxin, to initiate and support growth of callus and suspension cultures of several species is reported. Callus tissue of tobacco (Nicotiana tabacum L. var. WI-38), initiated and maintained on a medium with 2,4-dichlorophenoxyacetic acid (2,4-D), was transferred to and maintained on media supplemented with 25–500 M PAA as the only plant growth regulator (PGR). Optimal concentrations of PAA were determined for tobacco callus proliferation in the dark (250 M PAA) and with a 16-h light/8-h dark photoperiod (500 M PAA). Tobacco suspension cultures were maintained for over 28 transfers in media containing 20–40 M PAA as the sole PGR. When tobacco callus tissue maintained on PAA-supplemented media for over 18 months was transferred to liquid media containing kinetin, plantlets were regenerated. Callus of sunflower (Helianthus annuus L. var. Russian Mammoth) proliferated on media containing PAA at 5–250 M as the sole PGR. Similar PAA concentrations inhibited normal development and promoted callus formation in tobacco and pea (Pisum sativum L. vars. common, Frogel, and Frimas) epicotyl tissue. PAA as the sole PGR did not support the growth of soybean (Glycine max (L.) Merrill var. Fiskeby) callus or suspension cultures. Chickpea (Cicer arietinum L. var. UC-5) and lentil (Lens culinaris Medic. var. Laird) callus cultures proliferated on media containing 25–500 M PAA, but habituation of the cultures was common. PAA was not toxic to tobacco, chickpea, and lentil tissues at levels as high as 500 M.Paper No. 88514 of the Journal Series of the Idaho Agricultural Experiment Station, Moscow, Idaho, USA.     

Auxin activity of phenylacetic acid in tissue culture

The ability of phenylacetic acid (PAA), a naturally occurring auxin, to initiate and support growth of callus and suspension cultures of several species is reported. Callus tissue of tobacco (Nicotiana tabacum L. var. WI-38), initiated and maintained on a medium with 2,4-dichlorophenoxyacetic acid (2,4-D), was transferred to and maintained on media supplemented with 25–500 μM PAA as the only plant growth regulator (PGR). Optimal concentrations of PAA were determined for tobacco callus proliferation in the dark (250 μM PAA) and with a 16-h light/8-h dark photoperiod (500 μM PAA). Tobacco suspension cultures were maintained for over 28 transfers in media containing 20–40 μM PAA as the sole PGR. When tobacco callus tissue maintained on PAA-supplemented media for over 18 months was transferred to liquid media containing kinetin, plantlets were regenerated. Callus of sunflower (Helianthus annuus L. var. Russian Mammoth) proliferated on media containing PAA at 5–250 μM as the sole PGR. Similar PAA concentrations inhibited normal development and promoted callus formation in tobacco and pea (Pisum sativum L. vars. common, Frogel, and Frimas) epicotyl tissue. PAA as the sole PGR did not support the growth of soybean (Glycine max (L.) Merrill var. Fiskeby) callus or suspension cultures. Chickpea (Cicer arietinum L. var. UC-5) and lentil (Lens culinaris Medic. var. Laird) callus cultures proliferated on media containing 25–500 μM PAA, but habituation of the cultures was common. PAA was not toxic to tobacco, chickpea, and lentil tissues at levels as high as 500 μM.