Double immunocytochemical staining for the in situ study of allotype distribution during an anti-trinitrophenyl immune response in chimeric rabbits

After incubation of tissue sections with anti-allotype-enzyme conjugates, the localization of immunoglobulin-allotype-bearing cells in the lymphoid tissues of conventional and chimeric rabbits could be established. The use of anti-allotype sera bearing distinct enzyme labels allowed simultaneous recognition of B cells producing immunoglobulin of one or the other parental types in heterozygous rabbits, or of B cells from the donor and recipient in chimeras. After immunization of chimeric rabbits with trinitrophenyl-keyhole limpet hemocyanin, anti-trinitrophenyl antibody-forming cells could be demonstrated through the use of a trinitrophenyl-alkaline phosphatase conjugate. Simultaneous incubation of sections with this reagent and with horseradish peroxidase coupled to (donor or recipient) anti-allotype sera made possible the determination of the origin (donor or recipient) of the antibody-forming cells. In agreement with the results of plaque assays and analyses of serum antibodies, all the anti-TNP producing cells were of donor origin when the chimeras had been created through injection of spleen or lymph node cells from trinitrophenyl primed donors. With this study we introduce a simple, direct method for the simultaneous identification of cells that produce antibody of a given allotype and a given specificity, applicable to appropriate studies in heterozygous or chimeric rabbits. The procedure has various advantages over previously reported methods.     

Fusion of bacterial spheroplasts by electric fields

Spheroplasts of Escherichia coli or Salmonella typhimurium were found to fuse in an electric field. We employed the fusion method developed by Zimmermann and Scheurich (1981): Close membrane contact between cells is established by dielectrophoresis (formation of chains of cells by an a.c. field), then membrane fusion is induced by the application of short pulses of direct current. Under optimum conditions the fusion yield was routinely 90%. Fusable spheroplasts were obtained by first growing filamentous bacteria in the presence of cephalexin, then converting these to spheroplasts by the use of lysozyme. The fusion products were viable and regenerated to the regular bacterial form. Fusion of genetically different spheroplasts resulted in strains of bacteria possessing a combination of genetic markers. Fusion could not be achieved with spheroplasts obtained by growing the cells in the presence of penicillin or by using lysozyme on bacteria of usual size.     

Immunocytochemical demonstration of tissue-type plasminogen activator in endocrine cells of the rat pituitary gland

We immunocytochemically stained rat pituitary glands using antibodies against plasminogen activators of the tissue type (t-PA) and the urokinase type (u-PA). A large population of endocrine cells in the anterior lobe of the gland displayed intense cytoplasmic immunoreactivity with anti-t-PA. In some areas of the intermediate lobe we found a weak staining, and we observed weakly staining granular structures in the posterior lobe. Controls included absorption of the antibodies with highly purified t-PA. In addition, SDS PAGE followed by immunoblotting of pituitary gland extracts revealed only one band with an electrophoretic mobility similar to that of t-PA when stained with anti-t-PA IgG. No u-PA immunoreactivity was detected in the rat pituitary gland. Sequential staining experiments using antibodies against growth hormone and t-PA demonstrated that the t-PA-immunoreactive cells constitute a large subpopulation of the growth hormone-containing cells. These findings represent the first direct evidence for the presence of t-PA in cell types other than endothelial cells in the intact normal organism. In this article we discuss the implications of the results for a possible role of t-PA in the posttranslational processing of prohormones.     

Photoacoustic and fluorescence measurements of the chilling response and their relationship to carbon dioxide uptake in tomato plants

The response of tomato plants to various chilling treatments was studied using two approaches for the measurement of photosynthetic activity. One involved the use of a portable fluorometer for the measurement of in-vivo chlorophyll fluorescence, while the other employed a newly introduced photoacoustic system which allowed changes in oxygen evolution to be followed in a leaf disc. A strong correlation was found between results obtained by each system and those obtained by a conventional open gas-exchange system for the determination of CO₂ uptake. Both systems of measurements could readily distinguish between the effects of chilling in the dark (at 3° C for 18 h) and chilling at high photon flux density (2000 mol m^-2 s^-1 for 5h at 5° C). Chilling in the dark had practically no effect on the quantum yield of oxygen evolution, chlorophyll fluorescence or CO₂ uptake, while chilling at excessively high photon flux density resulted in a sharp reduction (50–70%) in the quantum yields obtained. The results support the view that photosystem II cannot be the primary site of damage by chilling in the dark, although it is significantly affected by chilling at high light intensity.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PA photoacoustic - PFD photon flux density - PSII photosystem II     

The parasitoids of the aleyrodid Bemisia tabaci in Israel: development, host preference and discrimination of the aphelinid wasp Eretmocerus mundus

Developmental history and behavior of Eretmocerus mundus Mercet, a parasitoid of Bemisia tabaci was studied at 25°C. The eggs may be laid under all four nymphal instars but not under the pupa. Yet the second and third instars are preferred. The egg hatches only under the fourth instar or the pupa. Developmental medians at 25°C are: Instar I-2.5, II-4, II-4, prepupa-2 and pupa 8 days. When ovipositing, the female stands at an angle of 90° to the host, with wings raised and inserts the ovipositor under the whitefly nymph. The egg is laid close to the insertion point of the whitefly's proboscis into the leaf. After oviposition, the female apparently marks the host while drumming on it with her hind legs. She distinguishes already parasitized hosts from unparasitized ones and refrains from laying under the former. Discrimination is accomplished after antennal drumming only.

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Les parasitoïdes de Bemisia tabaci (aleyrodidae) en Israel: développement, ponte et sélection des hôtes ches Eretmocerus mundus (aphelinidae)

Résumé Le développment et le comportement de E. mundus, parasitoïde de B. tabaci, ont été étudiés à 25°C. Les oeufs sont pondus sous les quatre stades larvaires (les deuxième et troizième sont préférés) mais pas sous les nymphes. Les oeufs n'éclosent que sous les larves du quatrième stade ou les nymphes. Les temps de développement médiaux sont à 25°, les suivants: stade I: 2,5j; stade II: 4j; stade III: 4j et nymphe 8j. Pendant la ponte, la femelle est à 90° sur son hôte, les ailes dressées, et insère sa tarière sous la larve d'aleurode. L'oeuf est déposé près du point d'insertion de la trompe dans la feuille. Après l'émission, la femelle marque apparemment son hôte pendant qu'elle tambourine avec ses pattes postérieures. Elle distingue les hôtes parasités ou non, et limite sa ponte dans les premiers. La sélection est effectuée seulement après tambourinage antennaire.

     

Modes of endocytosis of latex particles in sinusoidal endothelial and Kupffer cells of normal and perfused rat liver

The endocytosis of latex particles (0.33, 0.46 and 0.80 micron in diameter) in the sinusoidal endothelial and Kupffer cells of the rat liver was studied electron microscopically. When the liver was perfused with serum-free oxygenated Krebs Ringer bicarbonate, latex particles of all three sizes were taken up by the endothelial cells. After a 10-min perfusion, particles were incorporated by the luminal cell surface of the perikarya or of the thick portion of the endothelial cells. A large patch of bristle coat was surrounding the ingested particle. The number of ingested particles in the endothelial cells, however, was much less than in the Kupffer cells. In in vivo experiments, no endocytosis of the latex particles was observed in the endothelial cells. In the Kupffer cells, particles were engulfed by the ruffled membranes or sank into the cytoplasm without a large patch of the bristle coat both in the perfusion system and in vivo. These observations show that at least 0.80 micron latex particles are taken up by the bristle-coated membranes in the sinusoidal endothelial cells of the perfused liver. The endocytic mechanism for latex particles in the endothelial cells is different from that of the Kupffer cells.     

Reversible addition of bisulfite buffer to the cytidine ring system

The rate constants for the reversible addition of protons and sulfite to the 5,6 double bond of cytidine and 3-methylcytidine have been spectrophotometrically measured under conditions (25°C, μ = 1.0 ) where the deamination of 5,6-dihydrocytidine-6-sulfonate is minimal. Both the addition and the elimination of sulfite from the ring system are subject to general catalysis of proton transfer. For the reaction in either direction, plots of the pseudo-firstorder rate constants against increasing buffer concentration are biphasic and indicative of at least a two-step reaction pathway with both steps being subject to general acid-base catalysis. Kinetic hydrogen-deuterium isotope effects were measured for both buffer-catalyzed steps of sulfite elimination from 3-methyl-5,6-dihydrocytidine-6-sulfonate and sulfite addition to 3-methylcytidine. Both H₂O and D₂O were used as solvent. For both the addition and the elimination of SO₃²⁻ values of k₂^H/k₂^D were 6.3–7.1 and 2.3–2.6 at low and high imidazole buffer concentration, respectively. The large isotope effects values in the range of 6–7 can be attributed to rate-determining proton transfer to carbon-5 of the cytidine ring system. The smaller values are more likely caused by proton transfer to a electronegative atom such as the oxygen on carbon-2 of the cytidine ring. The equilibrium constants for bisulfite buffer addition to 3-methylcytidine and cytidine at 25°C, μ = 1.0 , pH 7.2, are 10.2 and 1.3 ⁻¹, respectively.     

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     

Orientation in a desert lizard (Uma notata): time-compensated compass movement and polarotaxis

Summary The diurnal escape response of fringetoed lizards (Uma notata) startled by predators demonstrates clear directional orientation not likely to depend on local landmarks in the shifting sands of their desert environment. Evidence that celestial orientation is involved in this behavior has been sought in the present experiments by testing the effects of (1) phase shifting the animal's internal clock by 6 h and (2) by training the lizards to seek shelter while exposed to natural polarization patterns. In the first case, 90° shifts in escape direction were demonstrated in outdoor tests, as expected if a time-compensated sun or sky polarized light compass is involved. In the second instance, significant bimodale-vector dependent orientation was found under an overhead polarizing light filter but this was only evident when the response data were transposed to match the zenithe-vector rotation dependent on the sun's apparent movement through the sky. This extends to reptiles the capacity to utilize overheade-vector directions as a time-compensated sky compass. The sensory site of this discrimination and the relative roles of sun and sky polarization in nature remain to be discovered.