Identification of Strychnine Binding Sites in the Rat Retina

Abstract: [³H]Strychnine specifically binds to membrane fractions isolated from rat retinae. The binding is saturable, with an apparent dissociation constant, K _D, of 14.3 × 10⁻⁹ M and 205 fmol bound/mg protein. Specific binding is time-dependent and proportional to protein concentration. Glycine and taurine are equally potent inhibitors of [³H]strychnine binding ( K _i= 4 × 10⁻⁵ M); no other amino acids endogenously present in the retina inhibited [³H]strychnine binding.     

GANGLIOSIDE COMPOSITION AND BIOSYNTHESIS IN CULTURED CELLS DERIVED FROM CNS

Abstract— Cultured mouse neuroblastoma cells (clone N18) contained a homologous series of gangliosides, G_M3, G_M2, G_M1 and G_D1a; the total lipid bound sialic acid (LBSA) was 3.3 nmol per mg of protein, of which G_D1a comprised two-thirds. In contrast, neonatal hamster astrocytes (clone NN) and human glioblastoma cells (Cox clone) contained mainly G_M3, which represented 95% of the 2 nmol of LBSA per mg protein in these cells. When the cells were grown in the presence of [¹⁴C]galactose, label was incorporated into all of the gangliosides isolated from the cells. The labeling pattern corresponded to the ganglioside composition of the cell lines; G_D1a was more extensively labeled in N18 cells and G_M3 was the major labeled ganglioside extracted from glial cells. In addition to in rivo biosynthesis, in vitro synthesis of gangliosides was also determined. The activities of five glycosyltransferases of the ganglioside biosynthetic pathway were measured in homogenates of the three cell lines. The neuroblastoma cells contained all five enzyme activities whereas the two glial cell lines were deficient in UDP- N -acetylgalactosamine: G_M3 N -acetylgalactosaminyltransferase activity, which catalyzes the synthesis of G_M2 from G_M3. The results indicated that cells of neuronal origin contain the more complex gangliosides associated with CNS and the requisite biosynthetic enzymes and that cells of glial origin are missing these complex gangliosides and the key glycosyltransferase required for their synthesis.     

Effects of Conformationally Restrained Analogues of Serotonin on Its Uptake and Binding in Rat Brain

Abstract: Two series of serotonin analogues, in which the side chain amino group is constrained in the gauche or trans conformation, were utilized to study the preferred conformation of serotonin for interaction with two different neuronal sites. 6-Hydroxytetrahydro-β-carboline and 6-hydroxy-3-aminotetrahydrocarbazole were found to be potent inhibitors of serotonin uptake into hypothalamic synaptosomes, with IC₅₀ values of 0.13 μM for each analogue. The type of inhibition, as determined by Dixon plots, was found to be competitive, with K_i's of 3.0 × 10⁻⁸ M and 4.6 × 10⁻⁸ M for the β-carboline and carbazole derivatives, respectively. Methoxylation or lack of a hydroxy group at the 6 position of the carbazole derivative did not alter inhibitory potency, while methoxy or benzyloxy substitution decreased potency 22- to 326-fold. The serotonin analogues were 20 to 30 times less potent in inhibiting the synaptosomal transport of the catecholamines. With regard to [³H]serotonin binding to membranes obtained from brain homogenates, both analogues exhibited poor affinity compared with the transmitter. However, the β-carboline derivative was three times as potent as the carbazole analogue. These findings and earlier ones with regard to the effect of the serotonin analogues on brain monoamine oxidase activity support the idea that serotonin analogues interact differentially with the three different serotonergic sites examined.     

Muscarinic Binding of Pial Vessels and Arachnoid Membrane

Abstract: The muscarinic sites in arachnoid and pial vessels were compared by analysis of the binding of quinuclidinyl benzilate (QNB) to membrane preparations. Saturation analysis indicated that the process was saturable, high affinity, and related to protein concentration in both structures. Although the affinities in the two structures [ K _D= 0.039 (arachnoid) and 0.097 n M (pial vessels)] were similar, the arachnoid had ∼ 10-fold more binding sites ( B _max= 2,100 fmol/mg of protein) than the pial vessels ( B _max= 250 fmol/ mg of protein). This difference was found in both bovine and porcine fractions. Pharmacological analysis of [³H]-QNB displacement by muscarinic and nonmuscarinic ligands gave the typical pattern of muscarinic receptors in both structures. Inhibition of binding to pial vessels by the M, antagonist pirenzepine revealed only one low-affinity site ( K _i= 7.8 × 10⁻⁷ M ), whereas, the arachnoid had a small proportion (21%) of high-affinity sites ( K _i= 2.2 × 10⁻⁹ M ) associated with low-affinity sites ( K _i= 5.50 × 10⁻⁷ M ). It is concluded that muscarinic-mediated effects that do not involve the M, subtype are induced in bovine pial vessels by a relatively low concentration of binding sites. The high content of muscarinic binding sites and their diversity in the arachnoid suggest a functional role for muscarinic cholinergic receptors in this structure.     

Effects of pH on the binding of Clostridium botulinum type E derivative toxin to gangliosides and phospholipids

Abstract Clostridium botulinum type E derivative toxin directly bound to gangliosides G_T1b, G_D1a, and G_Q1b but not to G_M1 or G_D1b at pH 5.0 or above, At the same pH values, it bound to negatively charged phospholipids but not to noncharged ones. At pH 4.0, it bound to any of gangliosides and phospholipids including G_M1, G_D1a, and non-charged phospholipids. It bound to ceramide, a hydrophobic component of ganglioside and also to sphingomyelin, a phospholipid containing a ceramide moiety, only at pH 4.0. It bound to ceramide and sphingomyelin less firmly than to other phospholipids at pH 4.0. We assume that botulinum toxin adheres to the neural cell surface mainly by sialic acid-specific and charge-dependent binding possibly aided by nonspecific hydrophobic(toxin)-hydrophobic(lipids, mainly phospholipids) interaction.     

Myelin Gangliosides in Developing Rats: The Influence of Maternal Ethanol Consumption

Abstract: The present study examined myelin gangliosides in the developing offspring of rats that were pair-fed control or ethanol liquid diets prior to and during gestation. Between 17 and 31 days of age, we observed an increase in the proportion of G_M1 in myelin (from 15% to 38% of ganglioside sialic acid) and a decrease in the proportion of G_T1b (from 26% to 4%). G_M4 was detected at all ages examined. Between 17 and 31 days of age, there was an increase in the proportion of N -acetylman-nosamine-derived radioactivity associated with G_M1 (from 16% to 22%) and G_M4 (from 5% to 13%), and a decrease in that associated with G_T1b (from 24% to 4%). Small, but sygnificant (p < 0.05), developmentally related differences were found in G_D2 and G_D3. Detection of G_M4 in myelin of young rats in the present study appears to depend on the use of nonpartitioning methods of ganglioside extraction. Although the distribution of myelin gangliosides and radioactivity was near-normal in ethanol-treated pups, there was a consistent decrease in the proportion and radioactivity associated with the major myelin ganglioside, G_M1.     

Quinuclidinyl Benzilate Binding in House Fly Heads and Rat Brain

Abstract: House fly heads contain a binding site for 3-quinuclidinyl benzilate (QNB) that is quite similar in pharmacology to the muscarinic acetylcholine receptor of vertebrate tissues. The house fly site binds [³H]QNB reversibly with a K _d of 260 PM and B_max of 1 pmol/g of heads from direct binding measurements. The K_d calculated from the ratio of the dissociation rate constant (2 × 10⁻⁴ sec⁻¹) to the association rate constant (2.5 × 10⁶ M⁻¹ Sec⁻¹) was 80 pM. The house fly site binds (-)quinuclidinyl benzilate preferentially, as do classic muscarinic receptors. The binding is also sensitive to other muscarinic antagonists and agonists. Nicotinic and other drugs are no more effective on the house fly site than they are on the rat brain muscarinic receptor itself. These binding studies suggest that the house fly QNB binding site is a muscarinic receptor.     

The Binding of Cations to the Surfaces of Cells from Early Chick Blastoderms

Cells of the early chick blastoderms are either preparing for or undergoing regulated morphogenetic movements which culminate in the formation of a three-layered embryo. Information on the changes in the physical-chemical properties of cell surfaces may help in the understanding of this process. The binding of magnesium, manganese, strontium, barium and lanthanum to surfaces of early embryonic cells was estimated by the changes induced by these cations in the cells' electrophoretic mobilities (EPM). Cells show a positive EPM at concentrations of MgCl₂ and MnCl₂ at 3 × 10⁻² M while SrCl₂, and BaCl₂ were not able to reverse the cells' charge at concentrations up to 6 × 10⁻² M. CaCl₃ reversed the cells' EPM at concentrations as low as 5 × 10⁻³ M.
Our results suggest that the surfaces of early embryonic cells have a high affinity for Mg and Mn. This is indicated by a reversal of polarity which cannot be detected in cells of differentiating or adult tissues at the cation concentrations used in these experiments.     

THE EFFECT OF DEVELOPMENT ON THE GANGLIOSIDES OF RAT AND PIG BRAIN

Abstract— The ganglioside content of the forebrain, brain stem and cerebellum have been studied, in the rat at various ages from 1 day to 27 months, and in the pig at various ages from 93 days gestation to 30 months. Each part of the brain was analysed for total ganglioside NANA and for four major gangliosides (G_Ml, G_D1a, G_Dlb and G_T1 in the nomenclature of S vennerholm , 1963). In the rat forebrain, the concentration of ganglioside NANA rose rapidly between 1 and 21 days after birth, fell to 3 months and subsequently rose to a mature value at 6 months. In the rat cerebellum, the peak concentration was reached at 2 months and the lower adult value at 9 months, whilst in the brain stern, the concentration rose more slowly and had a broad peak from 15 days to 2 months. Values are also given for the changes in the total amounts in each brain part. The changes in the concentrations and total amounts of ganglioside NANA, in the three parts of the pig brain were, on the whole, similar to those in rat brain except that the percentage distribution of the major gangliosides had almost attained the mature pattern at birth. In the forebrain of both species, the disialoganglioside, G_D1a, accounted for the highest percentage of the total gangliosides. The results are discussed with respect to their possible structural significance.     

Cellular localization of gangliosides in the developing mouse cerebellum: analysis using the weaver mutant

Abstract: The distribution of gangliosides was studied in the weaver ( wv/wv ) mutant mouse, where the vast majority of postmitotic granule cell neurons die prior to their differentiation. The wv mutation also shows a dosage effect, as granule cell migration is slowed or retarded in the + /wv heterozygotes. By correlating changes in ganglioside composition with the well-documented histological events that occur during cerebellar development in the normal (+/+), heterozygous ( +/wv ), and weaver ( wv/ wv ) mutant mice, information was obtained on the cellular localization and function of gangliosides. Ganglioside G_M1 may be enriched in granule cell growth cones and play an important role in neurite outgrowth. A striking accumulation of G_M1, which may result from altered metabolism, occurred in the adult wvlwv mice. G_D3 was heavily concentrated in undifferentiated granule cells, but was rapidly displaced by the more complex gangliosides during differentiation. G_D1a became enriched in granule cells during formation of synaptic and dendritic membranes, whereas G_T1a appeared enriched in Purkinje cell synaptic spines. A possible fucose-containing ganglioside was quantitated only in the wvlwv mice. Ganglioside G_T1b became enriched in granule cells during synaptogenesis, whereas G_Q1b became enriched in these cells after synaptogenesis. The concentrations of G_T1b and especially G_Q1b increased continuously with age. Our results provide further evidence for a differential cellular enrichment of gangliosides in the mouse cerebellum and also suggest that certain gangliosides may be differentially distributed within the membranes of these cells at various stages of development.     

Neurites and Somata of Sensory and Sympathetic Neurons in Culture Contain Multiple Species of Ganglio sides

Abstract: We have used tissue culture methods to study the capacity of sensory and sympathetic neurons to synthesize gangliosides. Under appropriate culture conditions, explants of dorsal root or superior cervical ganglia generate an extensive halo of ncurites, which is substantially free of contaminating cells. The cultures incorporate enough [³H]glucosamine into glycolipids to allow biochemical characterization. Gangliosides synthesized by the cells are extracted and freed of radioactive precursors and other lipids by column chromatography. Synthesized material comigrates on (thin-layer chromatograms with the gangliosides, G_Q, G_T, G_D1a, G^D1b, and G_M1, In addition, a substantial amount of unidentified labeled material migrates in a region between the mono- and disialo bands. The ganglioside profiles show that a similar spectrum of gangliosides is found on the neurites and somata of a particular class of neuron. Furthermore, the ganglioside compositions of the two types of neuron studied appear to be similar. We conclude that both sensory and adrenergic autonomic neurons synthesize gangliosides of each of the major classes and that representatives of each class are found in both somata and neurites. Dispersed cell cultures of the superior cervical ganglion synthesize a repertoire of gangliosides similar or identical to that of explant cultures.     

Detection of the response regulator AgrA in the cytosolic fraction of Staphylococcus aureus by monoclonal antibodies

Abstract The interaction of salivary lysozyme with the surface protein antigen (PAc) of Streptococcus mutans and the interaction of lysozyme with the pathogen were examined by ELISA using S. mutans MT8148 (PAc⁺) and the PAc-defective mutant EM-2 (PAc⁻). The lysozyme clearly bound to the S. mutans wild type but not to the S. mutans mutant. Furthermore, lysozyme bound directly in the fluid phase to the rPAc, of which the binding kinetics were determined ( K _on= 3.63 ± 0.04 × 10³M⁻¹ s ⁻¹, K _off= 1.72 ± 0.04 × 10⁻⁵s⁻¹ and K_on / K_off= 2.11 × 10⁸M⁻¹) using surface plasmon resonance. The kinetics of both association and dissociation were relatively slow. In addition, anti-lysozyme antibody significantly inhibited the binding of salivary components to the rPAc. The present findings indicate that lysozyme is one of the major salivary components interacting with S. mutans PAc.     

Retinal Gangliosides in RCS Mutant Rats

Abstract: The distribution of retinal gangliosides was studied in normal and mutant rats with retinal dystrophy at 30 and 180 days of age. The loss of photoreceptor cells in the retinal dystrophic RCS rats was not associated with a significant reduction in the relative distribution of any of the major retinal gangliosides. The loss of photoreceptors, however, caused a marked increase in total retinal ganglioside concentration. These findings suggest that photoreceptor cells contain a low concentration of gangliosides and that no major retinal ganglioside is localized or concentrated in these cells. The cellular localization and function of the most abundant retinal ganglioside, G_D3, is discussed.     

EFFECT OF DEVELOPMENT ON THE GANGLIOSIDES OF HUMAN BRAIN

Abstract— The ganglioside content and composition of brains from twenty-five human fetuses, three new-born babies and ten children, were studied. The ages ranged from 13 weeks gestation to 26 months postpartum. Each brain was divided into forebrain. cerebellum and brain stem. The concentration of total gangliosides rose to a plateau at different stages of development in the different parts, whereas the total amount reached a constant value at 9 months of age in each part. The developmental profile of individual gangliosides differed in the different parts of the brain. Thus, in the forebrain G_D1a. and in the cerebellum G_D1a rose to become the major gangliosides. The brain stem showed little change in its ganglioside pattern during the developmental period studied. The possible significance of these charges in the gangliosides during development is discussed.     

Multisite Interactions Between Pb2+ and Protein Kinase C and Its Role in Norepinephrine Release from Bovine Adrenal Chromaffin Cells

Abstract: We investigated the interaction between Pb²⁺ and protein kinase C (PKC) in the Pb²⁺-induced release of norepinephrine (NE) from permeabilized adrenal chromaffin cells. Our analysis of endogenous PKC activity in permeabilized cells suggests that Pb²⁺ interacts with the adrenal enzyme at multiple sites. Pb²⁺ activates the enzyme through high-affinity ( K _A(Pb) = 2.4 × 10⁻¹² M ) interactions and inhibits the enzyme by competitive and noncompetitive interactions with nanomolar-( K _i = 7.1 × 10⁻⁹ M ) and micromolar- ( K '_i = 2.8 × 10⁻⁷ M ) affinity sites, respectively. Activation of PKC by 12- O -tetradecanoylphorbol 13-acetate (TPA) in Ca²⁺-deficient, Pb²⁺-containing medium, enhances the Pb²⁺-induced NE release from permeabilized chromaffin cells by lowering the concentration of Pb²⁺ required for half-maximal activation of the secretory response from 7.5 × 10⁻¹⁰ to 5.7 × 10⁻¹¹ M . The PKC inhibitors staurosporine and pseudosubstrate PKC (19–36) abolish the effect of TPA without affecting the Pb²⁺-induced secretion in the absence of TPA. These results indicate that (a) Pb²⁺ is a partial agonist of PKC, capable of both activating and inhibiting the enzyme and (b) synergistic activation of PKC by TPA and Pb²⁺ results in increased sensitivity of exocytosis to Pb²⁺ but is not obligatory for Pb²⁺-triggered secretion.     

Emission rate of amino acids and ammonia and their role in olfactory preference behaviour of juvenile Arctic charr, Salvelinus alpinus (L.)

The behaviour of juvenile Arctic charr, Salvelinus alpinus (L.) , to an abrupt concentration step of L-amino acids, L-alanine and ammonium chloride was studied by fluviarium technique. The emission rates of these substances were studied. Juvenile Arctic charr emit 8.0 × 10⁻⁴ mol total ammonia-N kg⁻¹ h⁻¹ and 3.3 × I0⁻⁵ mol amino acids kg⁻¹ h⁻¹. In behaviour tests the charr avoided 5.6x 10⁻⁶and 5.6 × 10⁻⁷ M ammonium chloride. The 17 L-amino acid mixture, ranked as observed in the analysis of emission, was avoided at 4.6 × 10⁻⁷ M, while 100 times dilution of this value gave neither avoidance nor attraction. The charr avoided L-alanine tested alone at the concentration of 4.6 × 10 ⁻⁷ M. Anosmic charr showed neither avoidance nor attraction to the mixture of 17 amino acids tested at 4.6 × 10⁻⁷ M. The results indicate that ammonia as well as emitted amino acids are not responsible for the olfactory mediated attraction to conspecific odour shown earlier in Arctic charr. On the contrary, these substances may have a negative effect by reducing the strength of attraction.     

THE BINDING OF 5-HYDROXYTRYPTAMINE TO BUTANOL EXTRACTS OF RAT BRAIN STEM

Abstract— When butanol-water extracts of rat brain stem were incubated with [³H]5-HT, (5 × 10⁻⁷ m ), and the components resolved by chromatography on LH₂₀ Sephadex, a peak representing approximately 70% of the eluted radioactivity was found in chloroform-methanol 4:1. The peak was not found in identically prepared extracts from rat diaphragm, neither was a similar peak found when brain extracts were incubated with [¹⁴C]ACh (10⁻⁶ m ), suggesting a degree of selectivity. Binding was not saturated at concentrations of 5 × 10⁻⁵ m -5-HT. The binding was highly sensitive to the presence of water, requiring about 15% (v/v) for optimum binding. The implications of these findings are discussed in terms of a possible '5-HT receptor'.     

Flooding, gas exchange and hydraulic root conductivity of highbush blueberry

Highbush blueberry plants ( Vaccinium corymbosum L. cv. Bluecrop) growing in containers were flooded in the laboratory for various durations to determine the effect of flooding on carbon assimilation, photosynthetic response to varying CO₂ and O₂ concentrations and apparent quantum yield as measured in an open flow gas analysis system. Hydraulic conductivity of the root was also measured using a pressure chamber. Root conductivity was lower and the effect of increasing CO₂ levels on carbon assimilation less for flooded than unflooded plants after short-(i-2 days), intermediate-(10–14 days) and long-term (35–40 days) flooding. A reduction in O₂ levels surrounding the leaves from 21 to 2% for unflooded plants increased carbon assimilation by 33% and carboxylation efficiency from 0.012 to 0.021 mol CO₂ fixed (mol CO₂)⁻¹. Carboxylation efficiency of flooded plants, however, was unaffected by a decrease in percentage O₂, averaging 0.005 mol CO₂ fixed (mol CO₂)⁻¹. Apparent quantum yield decreased from 2.2 × 10⁻¹ mol of CO₂ fixed (mol light)⁻¹ for unflooded plants to 2.0 × 10⁻³ and 9.0 × 10⁻⁴ for intermediate- and long-term flooding durations, respectively. Shortterm flooding reduced carbon assimilation via a decrease in stomatal conductance, while longer flooding durations also decreased the carboxylation efficiency of the leaf.     

Characterization of ammonium and methylamine transport systems in phototrophic sulfur bacteria

Abstract Transport of ammonium and methylamine into the cells of green sulfur bacterium Chlorobium limicola and purple sulfur bacterium Thiocapsa roseopersicina is carried out by a common transport system. This system has (for C. limicola and T. roseopersicina , respectively) pH optimum 7.0 and 7.5; V _max 0.6 and 4.2 nmol min⁻¹ (mg protein)⁻¹; K_m 5.9 × 10⁻⁵ M and 1.3 × 10⁻⁵ M, and is capable of forming 120- and 600-fold methylamine gradients. The methylamine transport can be energized by the artificially imposed transmembrane K⁺ diffusive potential and is inhibited by tetraphenylphosphonium or valinomycin and K⁺. The data presented indicate that methylamine transport in both studied species is exclusively driven by the membrane potential gradient (ΔΨ).     

In Vivo Electrochemical Measurement of the Long-Lasting Release of Dopamine and Serotonin Induced by Intrastriatal Kainic Acid

Abstract: Intrastriatal injection of the glutamate agonist kainic acid (KA) in rats has been used to produce an animal model to investigate the mechanism of acetylcholine and GABA cell death associated with Huntington's disease. In the present study, the time course of low (10⁻⁵ M ) and high (5 × 10⁻³ M ) concentrations of KA on striatal dopamine and serotonin release was studied in freely moving rats by using in vivo voltammetry. The response to low concentrations of KA varied between animals, either increasing dopamine release during the injection or increasing dopamine and serotonin after the injection for an extended time, suggesting that 10⁻⁵ KA is near the threshold for KA toxicity in the striatum in rats. High concentrations of KA suppressed dopamine release during injection, with both dopamine and serotonin release increasing and remaining elevated for 1–4 and 7–21 days, respectively. KA-induced changes were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione and bicuculline increased the release of dopamine but not serotonin. These findings suggest that KA-induced changes in dopamine release resulted from a disinhibition of dopamine neurons due to KA-mediated toxicity of striatal GABA neurons. An alternate possibility is that the change in dopamine and serotonin release may have arisen from a functional modification or degeneration of presynaptic terminals.