Combined use of immunoperoxidase and radioimmunocytochemistry for double immunocytochemical labeling of neurons at light and electron microscopic level

Complexes formed by binding 125I- or 3H-labeled neuropeptides to one of the two binding sites of their specific antibodies allowed specific and sensitive labeling of various peptidergic neurons, which could be detected by classical autoradiographic methods. To visualize two neuronal antigens on the same material at both light and electron microscopic level, we used a new technique of double immunocytochemical labeling, combining immunoperoxidase and radioimmunocytochemistry. The main steps of the process included: (a) indirect labeling of the first antigen by its specific antibody and by a peroxidase-labeled Fab immunoglobulin fragment directed against the primary antibody; (b) direct labeling of the second antigen by a radiolabeled peptide-antibody complex; (c) revealing of the first label in the presence of peroxidase substrate; and (d) revealing of the second label by autoradiographic treatment of tissue sections. Compared with other known techniques of double immunostaining, this technique offers major advantages for combined visualization of two neuronal antigens at the electron microscopic level: (a) two neuron types can be labeled by a pre-embedding approach, allowing highly sensitive detection of neuronal antigens throughout the 50-microns thickness of vibratome sections; (b) two primary antibodies obtained in the same species can be used to label the two antigens without any risk of crossreactions between the two successive labelings; and (c) the two labels can easily be differentiated, even when they are co-localized within the same neuron structures. Application of this double immunostaining technique is illustrated by data obtained in rat hypothalamus concerning the relationships among a variety of identified neurons and the co-localization of different neuropeptides within the same neuron system.     

Determination of trace elements in aerosol samples by Instrumental Neutron Activation Analysis

Two nuclear techniques, Energy-Dispersive X-Ray Fluorescence Analysis (EDXRF) and Instrumental Neutron Activation Analysis (INAA), were used to analyze aerosol samples collected in the city of São Paulo, Brazil. Na, Cl, Mn, V, Al, Sm, Mo, W, La, As, Br, Sb, K, Ba, Se, Th, Cr, Rb, Ca, Fe, Ce, and Sc were determined by INAA, and Al, Si, P, S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Ni, Cu, Zn, Ga, As, Se, Br, Rb, Sr, Hg, and Pb were determined by EDXRF. A preliminary identification of the main source of the atmospheric aerosol was performed based on enrichment factor and correlation coefficient calculations.

     

Distribution of neuropeptide Y-like immunoreactive fibers in the cat thalamus

Neuropeptide Y-like immunoreactivity was studied in the thalamus of the cat using an indirect immunoperoxidase method. The densest network of immunoreactive fibers was observed in the nucleus (n.) paraventricularis anterior. In the anterior, intralaminar and midline thalamic nuclei, as well as in the n. geniculatum medialis, n. geniculatum lateralis, n. habenularis lateralis, n. medialis dorsalis, n. lateralis posterior and n. pulvinar a low density of neuropeptide Y-like immunoreactive fibers was observed. Neuropeptide Y-like fibers were totally absent in the n. ventralis lateralis, n. ventralis medialis, n. ventralis postero-medialis and n. ventralis postero-lateralis. In addition, neuropeptide Y-like perikarya were found in the n. parafascicularis, n. suprageniculatus, n. geniculatum lateralis ventralis, n. medialis dorsalis and n. lateralis posterior.     

Modeling competition between Laurencia obtusa (Ceramiales,Rhodophyta) and Hypnea spinella (Gigartinales,Rhodophyta) at Cabo Frio Island,Rio de Janeiro,Brazil

The Lotka-Volterra competition model was used to represent the interaction between Laurencia obtusa and Hypnea spinella. A new model that considers effects of competition on algal carrying capacity is suggested. To test the models, data from field experiments conducted in an intertidal region at Cabo Frio Island, Rio de Janeiro, Brazil, were used. Both models showed that Hypnea was a stronger competitor than Laurencia. The model of interaction through the carrying capacity showed a stable coexistence between the algal populations and better represented the experimental data.     

Trinuclear iridium and rhodium complexes: the solution to a puzzle involving the multiple coordination possibilities of 1,8-naphthyridine-2-one ligands

The multiple coordination possibilities of 1,8-naphthyridine-2-one (HOnapy) and 5,7-dimethyl-1,8-napthyridine-2-one (HOMe₂napy) ligands allow the synthesis of a variety of tri- di- and mononuclear complexes, showing fluxional behaviour and frequent exchange of the coordinated ML₂ fragments. Thus, reactions of [M₂(μ-OMe)₂(cod)₂] (cod = 1,5-cyclooctadiene) with HOnapy and HOMe₂napy yield the compounds of the general formula [M(μ-OR₂napy) (cod)]_n (M = Ir, R = Me (1a, 1b, H (2); M = Rh, R = Me (3a, 3b). They crystallise as inconvertible yellow (a) and purple/orange (b) forms and also show a puzzling behaviour in solution. X-ray diffraction studies on both forms (3a, 3b) and spectroscopic data reveal that the yellow forms are mononuclear complexes whilst the dark-coloured crystals contain dinuclear complexes. In solution, the nuclearity of the complexes depends on the solvent. In addition both types of complexes are fluxional. The mixed-ligand complexes [M₂(μ-OMe₂napy)₂(CO)₂(cod)] M = Ir (5), Rh (6) have been isolated and characterised; they are found to be intermediates in the synthesis of the trinuclear complexes [M₃(μ₃-OMe₂napy)₂(CO)₂(cod)₂]⁺ M = Rh (8), Ir (9). Reactions of [IrCl(CO)₂(NH₂-p-tolyl] with the complexes [Rh(μ-OR₂napy)(diolefin)]_n followed by addition of a poor donor anion is a general one-pot synthesis for the hetertrinuclear complexes [Rh₂Ir(μ₃-OR₂napy)₂(CO)₂(diolefin)₂]⁺ (R=Me, DIOLEFIN = cod (10), tetrafluorobenzo-barrelene (tfbb) (11), 2,5-norbornadiene (nbd) (12); R=H, DIOLEFIN=cod (13)). This synthesis follows a stepwise mechanism from the mononuclear to the trinuclear complexes in which mixed-ligand heterodinuclear complexes are involved as intermediates of the type [(diolefin)Rh(μ-OMe₂napy)₂Ir(CO)₂]. Heteronuclear complexes which possess the core [RhIr₂]³⁺, such as [RhIr₂(μ₃-OR₂napy)₂(CO)₂(cod)₂]BF₄ (R=Me (14), H (15)), result from the reaction of 1 or 2 with [Rh(CO)₂S_x]⁺ (S = solvent). The trinuclear complexes undergo two chemically reversible one-electron oxidation processes. The chemical oxidation of 10, 14 and 9 with silver salts gives the mixed-valence trinuclear radicals [Rh₂Ir(μ₃-OMe₂napy)₂(CO)₂(cod)₂]²⁺ (16), [RhIr₂(μ₃-OMe₂napy)₂(CO)₂(cod)₂]²⁺ (17) and [Ir₃(μ₃-OMe₂napy)₂(CO)₂(cod)₂]²⁺ (18), which have been isolated as the perchlorate and tetrafluoroborate salts. The EPR spectrum of 16 indicates that the unpaired electron is essentially in an orbital delocalised on the metals. The molecular structures of the complexes 3a, 3b, 6, 10b and 16a are described. Crystals of 3a are triclinic, P-1, with a = 9.7393(2), b = 14.0148(4), c = 16.0607(4) Å, α = 88.122(3), β = 83.924(3), γ = 87.038(3)°, Z = 4; 3b crystallises in the Pna2_i orthorhhombic space group, with a = 16.7541(3), B = 11.7500(8), c = 17.7508(7) Å, Z = 4; complex 6 is packed in the monoclinic space group P2_i/c, a = 9.6371(1), b = 11.8054(4), c = 27.2010(9) Å, β = 90.556(4)°, Z = 4; crystals of 10b are monoclinic, P2₁/n, with a = 17.546(7), b = 13.232(6), c = 17.437(8) Å, β = 106.18(1)°, Z = 4; crystals of 16a are triclinic, P-1, with a = 10.318(4), b = 12.562(6), C = 19.308(8) Å, α = 92.12(8), β = 97.65(9), γ = 90.68(5)°, Z = 2. The five different structures show the coordination versatility of the OMe₂napy molecule as ligand, which behaves as a N,N′-chelating (3a), bidentate N,O-donor (3b, 6), or as a tridentate N,N′,O-donor bridging ligand (10b, 16a).     

β2-Adrenoceptor activation by zinterol causes protein phosphorylation,contractile effects and relaxant effects through a cAMP pathway in human atrium

Evidence from ventricular preparations of cat, sheep, rat and dog suggests that both ₁-adrenoceptors (₁AR) and ₂-adrenoceptors (₂AR) mediate positive inotropic effects but that only ₁AR do it through activation of a cAMP pathway. On the other hand, our evidence has shown that both ₁ AR and ₂ AR hasten relaxation of isolated human myocardium consistent with a common cAMP pathway. We have now investigated in the isolated human right atrial appendage, a tissue whose -AR comprise around 2/3 of ₁AR and 1/3 of ₂AR, whether or not ₂AR-mediated effects occur via activation of a cAMP pathway. We carried out experiments on atria obtained from patients without advanced heart failure undergoing open heart surgery. To activate ₂AR, we used the ₂AR-selective ligand zinterol. Experiments were carried out on paced atrial strips (1 Hz) and tissue homogenates and membrane particles. Zinterol caused positive inotropic and lusitropic (i.e. reduction of t_1:2 of relaxation) effects with EC₅₀ values of 3 and 2 nM, respectively. The zinterol-evoked effects were unaffected by the AR-selective antagonist CGP 20712A (300 nM) but blocked surmountably by the ₂AR-selective antagonist ICI 118551 (50 nM) which reduced both EC₅₀ values to 1 M. Zinterol stimulated adenylyl cyclase activity with an EC₅₀ of 30 nM and intrinsic activity of 0.75 with respect to (–)-isoprenaline (600 M); the effects were resistant to blockade by CGP 20712A (300 nM) but antagonised surmountably by ICI 118551 (50 nM). Zinterol bound to membrane PAR labelled with (–)-[¹²⁵I] cyanopindolol with higher affinity for ₂AR than for - 1 AR; the binding to ₂AR but not to - BAR was reduced by GTPyS (10 M). In the presence of CGP 20712A (300 nM) (–)-isoprenaline (400 M); (to activate both ₁AR and ₂AR maximally) and zinterol (10 M); increased contractile force 3.4-fold and 2.5-fold respectively and reduced relaxation tut by 32% and 18% respectively. These effects of (–)-isoprenaline and zinterol were associated (5 min incubation) with phosphorylation (pmol P/mg supernatant protein) of troponin I and C-protein to values of 8.4 ± 2.0 vs 12.4 ± 2.3 and 10.1 ± 2.5 vs 8.6 ± 1.6 respectively. (–)-Isoprenaline and zinterol also caused phosphorylation of phospholamban (1.8 ± 0.3 vs 0.4 ± 0.1 pmol P/mg respectively) specifically at serine residues. We conclude that in human atrial myocardium activation of both ₁AR and ₂AR leads to cAMP-dependent phosphorylation of proteins involved in augmenting both contractility and relaxation.     

Fruit set in a deceptive orchid: The effect of flowering phenology,display size,and local floral abundance

We investigated the effect of flowering time, display size, and local floral density on fruit set in Tolumnia variegata, a pollination-limited orchid that offers no reward to its pollinator(s). During 1990, natural variation in flowering time, display size, and fruit set were monitored in 508 plants at one locality in Puerto Rico. The following season, orchid floral abundance per host tree (Randia aculeata) was manipulated to investigate its effect on fruit set. Four floral abundance treatments were established (700, 500, 300, and 100), each replicated four times. Flowering time was the most important trait affecting fruit set. The proportion of plants setting at least one fruit was significantly high early and late in the season, but low during the flowering peak. Thus, strong disruptive selection differential on flowering phenology was found. Display size had little effect on fruit set. A weak, but significant disruptive selection differential on display size was found. Orchid floral abundance per host tree had a significant effect on fruit set. Early in the season, T. variegata flowers with intermediate number of conspecific flowers exhibited a greater probability of setting fruit than those in host trees with fewer or more flowers. Our results show that flowering phenology may be evolutionarily unstable, possibly a consequence of the deception pollination system. Furthermore, a deception strategy would be relatively unsuccessful in populations where plants are found in either very dense or sparse patches.     

Molecular Cloning, Expression Pattern, and Chromosomal Localization of the Human Na–Cl Thiazide-Sensitive Cotransporter (SLC12A3)

Electrolyte homeostasis is maintained by several ion transport systems. Na–(K)–Cl cotransporters promote the electrically silent movement of chloride across the membrane in absorptive and secretory epithelia. Two kidney-specific Na–(K)–Cl cotransporter isoforms are known, so far, according to their sensitivity to specific inhibitors. We have cloned the human cDNA coding for the renal Na–Cl cotransporter selectively inhibited by the thiazide class of diuretic agents. The predicted protein sequence of 1021 amino acids (112 kDa) shows a structure common to the other members of the Na–(K)–Cl cotransporter family: a central region harboring 12 transmembrane domains and the 2 intracellular hydrophilic amino and carboxyl termini. The ex- pression pattern of the human Na–Cl thiazide-sensitive cotransporter (hTSC, HGMW-approved symbol SLC12A3) confirms the kidney specificity. hTSC has been mapped to human chromosome 16q13 by fluorescencein situhybridization. The cloning and characterization of hTSC now render it possible to study the involvement of this cotransport system in the pathogenesis of tubulopathies such as Gitelman syndrome.