Effect of spermidine on the conformation of bacteriophage MS2 RNA. Electron microscopy and computer modeling

The structure of single-stranded RNA from the bacteriophage MS2 has been examined by electron microscopy in the presence of the polyamine spermidine. The molecules are found in two alternate conformations. The first of these can be characterized as a cruciform structure composed of three large loops approximately 500 to 700 nucleotides in size. The interior of the molecule has extensive base-paired regions which connect distant regions of the molecule; the farthest being 2500 nucleotides apart. In the second conformation, the molecules appear rod-like. Two of the large loops disappear, and these regions form, instead, extensive long-range helices. Computer modeling has been employed to explore the base-pairing potential of the sequence of bacteriophage MS2 RNA. Double-stranded regions identified by electron microscopy are shown to occur in local G + C-rich stretches of the RNA. Detailed models have been calculated for two regions of long-range contact. One of these includes the ribosome-binding site for the viral coat protein gene. The results are discussed in the context of the known role of RNA structure in the regulation of viral gene expression.     

Augmentation of cholinergic-mediated amylase release by forskolin in mouse parotid gland

Cholinergic-mediated amylase release in mouse parotid acini was augmented by forskolin; the potency but not the maximal response to carbachol was altered. Amylase released by carbachol plus forskolin was dependent on extracellular calcium and was mimicked by the calcium ionophore, A23187 plus forskolin. Forskolin was also shown to enhance carbachol-stimulated 45Ca2+ uptake into isolated acini. Hydroxylamine, nitroprusside, and 8-bromo-c-GMP each in combination with forskolin mimicked the effects of carbachol plus forskolin on amylase release. In the presence of carbachol (10(-8)M) forskolin did not augment c-AMP levels. However, in the presence of carbachol (5 X 10(-7) M) or hydroxylamine (50 microM) forskolin did significantly augment c-AMP accumulation. These results suggest that calcium and c-GMP may mediate the augmentation of cholinergic-mediated amylase release by effects on c-AMP metabolism.     

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     

Neurite outgrowth traced by means of horseradish peroxidase inherited from neuronal ancestral cells in frog embryos

Outgrowing neurites in Xenopus embryos were labeled with horseradish peroxidase which had been injected into a single blastomere at the 32-cell stage and had been inherited by all the descendants, including neurons. Neurite outgrowth was traced from labeled trigeminal ganglion cells and most or all types of neurons present in the spinal cord at embryonic stages 20-30: primary motoneurons, commissural, dorsal longitudinal, ventral longitudinal, and Rohon-Beard neurons. All types of nerve fibers grew by the most direct pathway, apparently without errors of initial outgrowth, pathway selection, or target selection. An initial transient phase of outgrowth of filopodial processes from neuronal cell bodies and shafts of short neurites was observed which disappeared after further elongation of the neurites. The first pioneer fibers grew out from all types in a 2-hr period, from stage 20 to 22, and these fibers arrived at the targets within 3.5 hr after initial outgrowth. Additional fibers grew later in contact with the pioneers to form fascicles. Nerve fibers elongated without branching until they neared or contacted their targets. The rate of elongation at 20 degrees C was 30-75 micron/hr. The rapid, unbranched, error-free initial outgrowth and elongation of neurites to their targets is discussed in relation to theories of development of nerve pathways.     

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     

Migration of peripheral T and B cells into the thymus of aging (NZB × SJL)F1 female mice

Aging NZB × SJL (NS) female mice provide a unique model of thymopathology characterized by the intrathymic accumulation of large numbers of mature T and B cells. The purpose of the present work was to examine the possibility that this phenomenon results from the invasion of the thymus by cells from the periphery. Lymphoid cells labeled with chromium-51 or indium-111 were injected into syngeneic recipients to study their patterns of in vivo migration. Lymph node (LN) or spleen cells were found to localize significantly (1–2% of injected radioactivity) into the thymus of 12-month-old NS females but not into that of young recipients or of old NS males. However, intrathymic localization of injected LN cells was observed in castrated NS males which exhibit the same thymopathology as NS females. Both radiolabeled T and B cells were found to enter the thymus of aged NS females but the latter cells about three times less efficiently than the former. Moreover, while thymocytes from young NS females were unable to recirculate to LN, those of old NS females showed increased LN-seeking capacity and part (1%) of them did migrate back into the thymus of old but not young NS females. In additional cell transfer experiments, the intrathymic migration of B cells into old NS females was further documented by using the antibody response to sheep erythrocytes as a tracer. Taken together, these observations indicate that the thymus of aging NS female mice is permeable to recirculating lymphocytes, suggesting that at least part of the mature T and B cells detected in this thymus, are migrants from the periphery.