Coexistence of Probe Conformations in Lipid Phases—A Polarized Fluorescence Microspectroscopy Study

Several well-established fluorescence methods depend on environment-sensitive probes that report about molecular properties of their local environment. For reliable interpretation of experiments, careful characterization of probes’ behavior is required. In this study, bleaching-corrected polarized fluorescence microspectroscopy with nanometer spectral peak position resolution was applied to characterize conformations of two alkyl chain-labeled 7-nitro-2-1,3-benzoxadiazol-4-yl phospholipids in three model membranes, representing the three main lipid phases. The combination of polarized and spectral detection revealed two main probe conformations with their preferential fluorophore dipole orientations roughly parallel and perpendicular to membrane normal. Their peak positions were separated by 2–6 nm because of different local polarities and depended on lipid environment. The relative populations of conformations, estimated by a numerical model, indicated a specific sensitivity of the two probes to molecular packing with cholesterol. The coexistence of probe conformations could be further exploited to investigate membrane organization below microscopy spatial resolution, such as lipid rafts. With the addition of polarized excitation or detection to any environment-sensitive fluorescence imaging technique, the conformational analysis can be directly applied to explore local membrane complexity.     

Electroencephalographic changes in the lateral septum complex following systemic administration of DES-TYR1 -α-endorphin,Des-Tyr1-γ-endorphin and haloperidol in rats

The influence of systemically administered Des-Tyr¹-α-endorphin (DTαE), Des-Tyr¹-γ-endorphin (DTγE) and haloperidol on electroencephalographic (EEG) activity of the lateral septum complex (LSC) and the frontal cortex was studied in male rats. DTαE (2 μg) significantly increased whereas DTγE (10 μg) significantly decreased the amounts of activity in the 5 Hz band. In addition, DTαE promoted production of 15 - 20 Hz activity, while DTγE decreased the amount of 20 - 30 Hz activity. EEG activity exhibited a marked variability which persisted throughout the recording session following administration of the peptides. Haloperidol markedly decreased the proportion of 10 - 15 Hz activity. The alterations in EEG of the frontal cortex were similar to those in LSC but less pronounced. The differences in the time course and frequency bands affected suggest that the effects of peptides and haloperidol on EEG activity of LSC are not mediated by the same mechanisms.     

Genetic interaction between Non-MHC T- and B-cell alloantigens in response to rous sarcomas in chickens

Chickens of Regional Poultry Research Laboratory (RPRL) inbred line 6₃ regress sarcomas induced by Bryan high-titer Rous sarcoma virus to a greater extent than chickens of line RPRL 100, although these lines are identical for the major histocompatibility B complex. They differ, however, at three independent autosomal loci: Ly-4 and Th-1 determine the surface alloantigens of partly overlapping subsets of T lymphocytes, and Bu-1 determines a surface alloantigen of B lymphocytes. The association of genotypes at these loci with quantitative variation in their ability to regress Rous sarcomas was tested in segregating F₄ generation progeny derived from crosses of lines 100 and 6₃. The Ly-4 and Bu-1 genotypes showed association with Rous sarcoma regression, but the Th-1 genotype did not. Chickens of the Ly-4 ^a/Ly-4 ^a, Bu-1 ^b/Bu-1 ^b and Ly-4 ^b/Ly-4 ^b, Bu-1 ^a/Bu-1 ^a genotypes had a significantly higher regressor ability than the other two double homozygous genotypes. These results indicate that higher regression is associated with (1) interaction between the Ly-4 and Bu-1 loci, and (2) complementation between either the line 6 Ly-4 ^a allele and the line 100 Bu-1 ^b allele, or the line 100 Ly-4 ^b allele and the line 6 Bu-1 ^a allele.     

Purified Fc epsilon R+ bone marrow and splenic non-B, non-T cells are highly enriched in the capacity to produce IL-4 in response to immobilized IgE, IgG2a, or ionomycin.

Non-B, non-T cells from spleen and bone marrow cells produce IL-4 in response to cross-linkage of high affinity receptors for Fc epsilon R or Fc gamma RII, and to treatment with calcium ionophores. Cells bearing high affinity Fc epsilon R constituted 1 to 2% of non-B, non-T cells of spleen and of total bone marrow cells from naive donors. In mice whose immune systems had been polyclonally activated by injection with anti-IgD antibodies or had been infected with Nippostrongylus brasiliensis larvae, the frequency of Fc epsilon R+ cells in splenic non-B, non-T cells was also 1 to 2% but in bone marrow from anti-IgD-injected mice donors the frequency was approximately 5%. Cell sorting experiments revealed that all of the capacity to produce IL-4 in response to immobilized IgE or IgG2a or to ionomycin was found in the Fc epsilon R+ fraction. Among the Fc epsilon R+ spleen cells from naive donors, the frequency of IL-4-producing cells was 1/20 to 1/40 whereas in mice that had been injected with anti-IgD or infected with N. brasiliensis, the frequency of IL-4 producing cells in the Fc epsilon R+ population was approximately 1/5.     

Rhizobium trifolii 0403 Is Capable of Growth in the Absence of Combined Nitrogen

Rhizobium trifolii 0403 was treated with 16.6 mM succinate and other nutrients and thereby induced to grow in nitrogen-free medium. The organism grew microaerophilically on either semisolid or liquid medium, fixing atmospheric nitrogen to meet metabolic needs. Nitrogen fixation was measured via ¹⁵N incorporation (18% ¹⁵N enrichment in 1.5 doublings) and acetylene reduction. Nitrogen-fixing cells had a K_m for acetylene of 0.07 atm (ca. 7.09 kPa), required about 3% oxygen for optimum growth in liquid medium, and showed a maximal specific activity of 5 nmol of acetylene reduced per min per mg of protein at 0.04 atm (ca. 4.05 kPa) of acetylene. The doubling time on N-free liquid medium ranged from 1 to 5 days, depending on oxygen tension, with an optimum temperature for growth of about 30°C. Nodulation of white clover by the cultures showing in vitro nitrogenase activity indicates that at least part of the population maintained identity with wild-type strain 0403.     

High Performance Liquid Chromatography of Molecular Species from Free Sterols and Sterylglycosides Isolated from Oat Leaves and Seeds

Free sterols and sterylglycosides (SG) from oat leaves and seedswere isolated by conventional thin layer chromatography (TLC)and subjected to high performance liquid chromatography (HPLC)for resolution of molecular species. Acylsterylglycosides, isolatedby TLC, were converted to SG by mild alkaline hydrolysis anddetermined as SG. Sterols and SG were injected onto the columnwithout any chemical treatment and the separated species weredetected at 200 nm. The separation of SG-species follows exactlythe separation of free sterols. Though gas liquid chromatography still is the method of choice,advantages of HPLC is to analyse directly the SG-species withouthydrolysis and derivatization as compared to GLC. After TLCthe sterol- and the SG-fraction are injected directly onto thecolumn. This is extremely important for labile sterylglycosidesor sterols, as demonstrated for the avenasterols. ¹ Preliminary reports have been presented on the "4. Arbeitstagung,Pflanzliche Lipide", October 7–8, 1983 in M?nster (FRG)and on the "6th International Symposium on the Structure, Functionand Metabolism of Plant Lipids", Neuchatel, Switzerland, July16–20, 1984. (Received November 12, 1984; Accepted January 14, 1985)     

Ascaris suum: protective immunity in pigs immunized with products from eggs and larvae

Parasite products were collected at three distinct phases of development of Ascaris suum, and their immunogenicity was determined after injection into rabbits and pigs. Products were derived from (1) the hatching fluid of infective eggs; (2) the conditioned medium of 2nd-stage larvae that developed to 3rd stage in vitro in defined medium; and (3) the conditioned medium of 3rd-stage larvae that developed to 4th stage in vitro in defined medium. Protein profiles from these three preparations, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were less complex than that of extracts from homogenized A. suum larvae. Hyperimmune rabbit antiserum raised against either egg products, 2nd- to 3rd-stage larval excretory-secretory products, or 3rd- to 4th-stage larval excretory-secretory products showed strong homologous reactions after immunoelectrophoresis, but relatively weak cross-reactions with the other preparations. A combined enteral immunization of pigs with egg products and parenteral immunization with the 2nd- to 3rd-stage larval excretory-secretory products, and 3rd- to 4th-stage larval excretory-secretory products induced antibody to each preparation and significant protective immunity to a challenge exposure with 10,000 A. suum eggs. However, a marked pathological response to larvae migrating in the liver after challenge exposure was also induced.     

Factors contributing to the in vitro development of Ascaris suum from second-stage larvae to mature adults

When a three-step roller culture system was used, second-stage larvae of Ascaris suum, artificially hatched from eggs, developed in high numbers to the fourth stage, and a few to young and mature adults. The culture system consisted of (1) Medium KW-2 supplemented with 10 mM L-cysteine for the first 4 days, and with 5 mM L-cysteine for the following 7 days; (2) followed by Medium API-18 for 7 days; and (3) thereafter, by Medium API-1 supplemented with hemin (bovine) at a concentration of 24 micrograms/m1. Cultures were gassed with 95% nitrogen-5% carbon dioxide for the first 4 days and 85% nitrogen-5% oxygen-10% carbon dioxide thereafter, and incubated at 39 C. Two mature females that produced unfertilized eggs and a mature male with spermatozoa were the most advanced stages attained. The mature females were obtained in 67 and 73 days; and the largest female measured 110 mm. The latter produced 1,356,000 unfertilized eggs, from days 67 to 125. The mature male was obtained in 80 days; it measured 77 mm long and had paired spicules that were 1.5 mm long. Development of A. suum in three other culture systems showed that deletion of Medium API-18 or its substitution by Medium KW-2 limited development to late fourth stage and early, young adults, respectively; and the use of Medium API-1 without hemin limited development to early fourth stage.