Measurements of pH in the apoplast of sunflower leaves by means of fluorescence

A method was elaborated by which the pH in leaf apoplast can be measured. The technique is based on the pH dependent fluorescence of 5-carboxyfluorescein (5-CF) or fluorescein isothiocyanate (FITC). The fluorescein isothiocyanate is coupled with a macromolecular dextran molecule (FITC-dextran). For eliminating the effect of the absolute dye concentration the dual excitation technique was applied. It was shown that the ratio of fluorescence excited by light of 491 nm and 463 nm was virtually independent of the concentration of 5-CF and that this fluorescence ratio was related to the pH. The plasmalemma is practically impermeable to FITC-dextran and in the test we carried out over a period of 6 h not the slightest indication was found that it may penetrate the plasma membrane. For 5-CF this cannot be ruled out completely. It is possible that at pH values below 4.5 it may penetrate biological membranes at low rates.
Experiments with leaves of sunflower ( Helianthus animus cv. Erika) perfused with 5-carboxyfluorescein and supplied with different nitrogen forms showed that NH⁺₄ application resulted in a decrease and NO⁺₃ application in an increase of the leaf apoplast pH. Leaf spraying with fasicoccin was followed by a pH decrease, while leaf spraying with the protonophores p -trifluoromethoxy carbonytcyanide phenylhydra-zon (FCCP) or nigericin resulted in neutral apoplastic pH. These results provide evidence that the method is well suited for measuring the response of the leaf apoplast pH to changing physiological conditions.     

Evidence for a driving role of ingrowing axons for the shifting of older retinal terminals in the tectum of fish

In amphibians and teleosts, retina and tectum grow incongruently. In order to maintain the retinotopy of the retinotectal projection, Gaze, Keating, and Chung (1974) postulated a shifting of terminals throughout growth. In order to test the possibility that ingrowing retinal fibers are the driving force for this shifting, we induced a permanent retinal projection into the ipsilateral tectum in juveniles of the cichlid fish Haplochromis burtoni. The surface of the tectum had increased (11–18 months later) 2.5–5.8 times, and the surface of the retina 8.6–14 times. Filling of ganglion cells with horseradish peroxidase (HRP) retrogradely from the tectum showed ipsilaterally regenerating ganglion cells only in the center of the retina. The position of ganglion cells indicated that the ipsilateral projection derived only from axotomized and regenerating retinal ganglion cells but not from those newly born. Ipsilaterally projecting retinal fibers showed terminals only in the rostral half of the tectum. Comparison of area of terminations of ipsilaterally projecting ganglion cells at various times after the crush provided no evidence for expansion or a shift into caudal tectal areas throughout the period of growth. These findings are compatible with the idea that newly ingrowing fibers induce older terminals to move caudally.     

Separation of dissociated thyroid follicular and parfollicular cells: association of serotonin binding protein with parafollicular cells

Parafollicular cells (PC) of the sheep thyroid gland are neural crest derivatives that synthesize and release the biogenic amine serotonin (5-HT) as well as the hormone calcitonin. The thyroid also contains a highly specific serotonin-binding protein (SBP). Separation of dissociated thyroid cells was done to study the cellular localization of SBP and to develop a means of isolating PC for study. Various methods were used to obtain an enriched and purified population of PC. Minced thyroid glands were enzymatically dissociated and the cells were layered on a Ficoll linear density gradient. Fractions obtained from the gradient were examined for cell number, viability, 5-HT concentration, SBP activity, and morphology by electron microscopy. One of the fractions was found to be enriched in PC. High levels of 5-HT and SBP were also found in this fraction, whereas these levels were low where the majority of cells were found. This PC-rich fraction, however, contained numerous follicular cells (FC); therefore, additional approaches to cell separation were used. FC can be stimulated in vitro with thyroid stimulating hormone (TSH) to become intensely phagocytic. When stimulated cells were incubated in the presence of silica microspheres, the FC engulfed the microspheres, which were toxic to them. PC did not become phagocytic and were unharmed by the microspheres. Suspended cells, after incubation with microspheres, were centrifuged on a discontinuous gradient, and a PC-rich fraction was obtained. Silica, however, interfered with analysis of SBP. Another method to take advantage of the phagocytic potential of FC was therefore used. TSH-stimulated cell suspensions were passed through a column of sepharose to which thyroglobulin had been coupled. Stimulated FC apparently adhered to the beads and were retained by the columns. Fractions eluting from the columns were greatly enriched with PC. These fractions contained high levels of 5-HT and SBP, and considerably reduced FC contamination was found by quantitative electron microscopy. It is concluded that SBP is localized to PC in the sheep thyroid. The idea that these cells resemble serotonergic neurons in their mechanisms of 5-HT storage is supported.     

Dermorphins, Opioid Peptides from Amphibian Skin, Act on Opioid Receptors of Mouse Neuroblastoma x Rat Glioma Hybrid Cells

Dermorphin and its Hyp6 analogue are opiate-like heptapeptides originally discovered in frog skin and characterized by the presence of a D-Ala2 residue in their sequence. They were assayed for their capacity to compete with [3H]Leu-enkephalin for binding to opioid receptors in membranes of neuroblastoma x glioma hybrid cells. In the presence of 7 nM-[3H]Leu-enkephalin, the concentrations at which they caused 50% inhibition of [3H]enkephalin binding (IC50 values) are 0.1 micro M and 0.3 micro M, respectively. In contrast, the synthetic L-Ala2-dermorphin shows very low affinity for the opioid receptors. In addition, like other opioid peptides, dermorphin and hyp6-dermorphin inhibit the elevation by prostaglandin E1 (PGE1) of the level of adenosine 3':5'-cyclic monophosphate (cyclic AMP) (IC50 values 0.2 micro M and 0.4 micro M, respectively). The inhibition is prevented by the opiate antagonist naloxone, L-Ala2-dermorphin is at least three orders of magnitude less potent in inhibiting the PGE1-evoked increase in the level of cyclic AMP. The results show that peptides with an amino acid sequence quite different from that of the enkephalins can bind to opioid receptors of the hybrid cells.     

19-hydroxycoronaridin und 19-hydroxyibogamin,zwei antibiotisch wirksame alkaloide vom ibogamin-typ

During the separation of an extract from Tabernaemontana glandulosa, small amounts of a new alkaloid were obtained for which the structure 19-hydroxycoronaridine was deduced. This new alkaloid and 19-hydroxyibogamine, which can be obtained from it, show marked antibiotic activity in the agar plate diffusion test.     

Retinal projections in European Salamandridae

Summary The retinal projections to the brain were studied in three species of European Salamandridae using anterograde transport of horseradish peroxidase and autoradiography. The results obtained were basically identical for all species and confirmed earlier findings on the fiber supply to the preoptic nucleus and the basal optic neuropil. In the anterior thalamus projections to three distinct terminal fields are clearly visible: (i) the diffusely stained corpus geniculatum thalamicum, (ii) the neuropil of Bellonci, pars lateralis, and (iii) a dorsomedial terminal field, the neuropil of Bellonci, pars medialis. Caudal to these terminal fields is an almost terminal-free region, the lateral neuropil. In the posterior thalamus a medial terminal field, the uncinate field, and a laterally located terminal field, the posterior thalamic neuropil, are distinguishable. The tectum opticum displays as many as four dense layers of retinofugal fibers and terminals in the rostral part and, in addition, a more densely stained strip of neuropil running from rostral to caudal over the tectum. The extent of ipsilateral fibers is greater than previously reported in other urodele species. They supply the medial and the lateral parts of the neuropil of Bellonci, the uncinate field, and reach the tectum opticum via the medial optic tract. Further, they form terminals in the innermost optic fiber layer throughout the rostral half of the ipsilateral tectum. A small proportion of ipsilateral fibers contributes very sparsely to all other thalamic terminal fields, leaving only the caudal part of the tectum and several layers of the rostral tectum completely free of a direct retinofugal fiber supply.     

Properties of protease-treated cytochrome c oxidase from beef heart

Summary About 45% of the protein can be removed from oxidized cytochrome c oxidase by treatment with proteolytic enzymes under a variety of conditions, leading to an increased heme to protein ratio.The principal spectroscopic parameters of cytochrome c oxidase are retained in the protease-treated enzyme.Of the overall catalytic activity 20% remained after digestion; the electron-transfer reactions were impaired but the affinity for cytochrome c appeared unchanged.Proteolysis resulted in removal of the hydrophobic subunit III and most of the smaller hydrophilic subunits, leaving a core, which basically consists of the two largest subunits I and II.The subunits I and/or II carry the prosthetic groups of the enzyme and at least one of the cytochrome c binding sites. The smaller subunits, however, are essential for optimal electron transfer and possibly have other functions as well.