l-Phenylalanyl-l-Glutamate-Stimulated, Chloride-Dependent Glutamate Binding Represents Glutamate Sequestration Mediated by an Exchange System

Stimulation of glutamate binding by the dipeptide L-phenylalanyl-L-glutamate (Phe-Glu) was inhibited by the peptidase inhibitor bestatin, suggesting that the stimulation was caused by glutamate liberated from the dipeptide and not by the dipeptide itself. It further suggests that this form of glutamate binding should be reinterpreted as glutamate sequestration and that stimulation of binding both by dipeptides and after preincubation with high concentrations of glutamate is likely to be due to counterflow accumulation. Several other criteria indicate that most of glutamate binding stimulated by chloride represents glutamate sequestration: Binding is reduced when the osmolarity of the incubation medium is increased, when membranes incubated with [3H]glutamate are lysed before filtration, and when membranes are made permeable by transient exposure to saponin. Moreover, dissociation of bound glutamate after a 100-fold dilution of the incubation medium is accelerated about 50 times by the addition of glutamate to the dilution medium. This result would be anomalous if glutamate were bound to a receptor site; it suggests instead that glutamate is transported in and out of membrane vesicles by a transport system that preferentially mediates exchange between internal and external glutamate. Glutamate binding contains a component of glutamate sequestration even when measured in the absence of chloride. Sequestration is adequately abolished only after treating membranes with detergents; even extensive lysis, sonication, and freezing/thawing may be insufficient.     

Purification and Properties of Rubrophilin: A Novel Brain Specific Membrane Polypeptide

Rubrophilin, a unique brain specific polypeptide, was purified to apparent homogeneity from microsomal fractions of bovine brains. The peptide stains pink with Coomassie Brilliant Blue R-250 (C.I. No. 42660) under specific conditions, has an apparent Mr of 53,000, and is acidic with an apparent pI of 4.9. The purification involves initial solubilization of delipidated microsomes in sodium dodecyl sulfate, followed by ammonium sulfate fractionation, reversed ammonium sulfate gradient elution from diatomaceous earth, gel filtration on polyacrylamide (Biogel P-200), gradient elution chromatography from hydroxylapatite, and reverse-phase chromatography from phenyl-Sepharose. A yield of about 5 mg of rubrophilin was obtained from 9 g of microsomal proteins. Amino acid analysis shows that rubrophilin contains only nine amino acids with residues/mol as follows: alanine (102), glutamic acid (97), lysine (65), proline (55), aspartic acid (48), glycine (44), serine (37), threonine (35), and valine (10). Cysteine, methionine, tryptophan, tyrosine, isoleucine, phenylalanine, histidine, and arginine could not be detected. Relative rubrophilin content of vertebrate brains was as follows: mammals greater than birds greater than reptiles greater than fishes. It is present in mouse retina and human neuroblastoma cell cultures but could not be detected in octopus optic lobe or in cultured C-6 rat glioma cells.     

Phosphatidylinositol Phosphodiesterase (Phospholipase C) Activity in the Pineal Gland: Characterization and Photoneural Regulation

Phosphatidylinositol phosphodiesterase (PL-C) appears to be a key element in the adrenergic regulation of pineal cyclic AMP levels. In the present study, the rat pineal enzyme was characterized using exogenous [3H]phosphatidylinositol (0.5 mM) as substrate. Half the enzyme activity was found in the cytosolic fraction, but the highest specific concentration was associated with the membrane fraction. Two pH optima (5.5 and 7.5) of enzyme activity were observed for the membrane fraction but only one in the cytosol fraction (pH 5.5). Enzyme activity in both fractions was Ca2+ dependent. In the case of the membrane protein in pH 7.5, the enzyme activity was sensitive to changes in Ca2+ in the 10-100 nM range. Addition of an equimolar concentration of phosphatidylinositol 4-phosphate nearly completely inhibited the hydrolysis of [3H]phosphatidylinositol; other phospholipids (1.0 mM) were less potent. This may reflect our present finding that [3H]phosphatidylinositol 4-phosphate is a better substrate than [3H]phosphatidylinositol for the enzyme. Stimulus deprivation (2 weeks of constant light or superior cervical ganglionectomy) reduced the cytosolic activity by 30% and had no effect on the membrane-associated enzyme.     

Evidence for a sex pheromone metabolizing cytochrome P-450 mono-oxygenase in the housefly

Direct evidence is presented for the role of a cytochrome P-450 monooxygenase (called mixed-function oxidase, or polysubstrate mono-oxygenase, PSMO) in the metabolism of the sex pheromone (Z)-9-tricosene to its corresponding epoxide and ketone in the housefly. A secondary alcohol, most likely an intermediate in the conversion of the alkene to the ketone, was also tentatively identified. The results of in vivo and in vitro experiments showed that the PSMO inhibitors, piperonyl butoxide (PB) and carbon monoxide, markedly inhibited the formation of epoxide and ketone from (9,10-³H) (Z)-9-tricosene. An examination of the relative rates of (Z)-9-tricosene metabolism showed that males exhibited a higher rate of metabolism than females with the antennae of males showing the highest activity of any tissue/organ examined. The major product from all tissues/organs was the epoxide. Data from experiments with subcellular fractions showed that the microsomal fraction had the majority of enzyme activity, which was strongly inhibited by PB and CO and required NADPH and O₂ for activity. A carbon monoxide difference spectrum with reduced cytochrome showed maximal absorbance at 450 nm and allowed quantification of the cytochrome P-450 in the microsomal fraction of 0.410-nmol cytochrome P-450 mg^?1 protein. Interaction of (Z)-9-tricosene with the cytochrome P-450 resulted in a type I spectrum, indicating that the pheromone binds to a hydrophobic site adjacent to the heme moiety of the oxidized cytochrome P-450.     

Naturally occurring monoterpenoids in needles of Picea abies (L.) Karst

Summary An analysis was made of the effects of different sampling and extraction techniques on the amounts and pattern of monoterpenoids isolated from needles of Norway spruce. The following isolation and analysis procedure was finally adopted: liquid nitrogen-cooled needles were pulverized by a microdismembrator, extracted with pentane overnight at 2°–3°C and concentrated to a volume not less than 3 ml/g fresh weight on a Vigreux column. The crude extract was injected splitless (with solvent split) onto a cold programmed temperature vaporized (PTV) precolumn of a gas chromatograph and the vaporizable compounds heated to a capillary column. This method was tested for production of artefacts and quantitative extraction and applied to needles of eleven 80-year-old spruce trees.     

Distinct phases of the fluorescence response of the lipophilic probe N-phenyl-1-naphthylamine in intact cells and membrane vesicles of Escherichia coli

The fluorescence of the lipophilic prbe N-phenyl-1-naphthylamine (NPN) bound to intact cells of Escherichia coli is quenched by the addition of glucose, succinate, -lactate, pyruvate, formate and glycerol. Partial recovery of fluorescence occurs on anaerobiosis. Use of mutants with defects in the ATP synthase or the respiratory chain show that quenching of fluorescence may be energized either by ATP hydrolysis or by substrate oxidation through the respiratory chain. Permeabilization of the outer membrane by treatment of intact cells with EDTA, or use of a mutant with an outer membrane permeable to lipophilic substances, results in a more rapid binding of NPN and in a decrease in quenching observed on substrate addition. NPN binds rapidly to everted membrane vesicles, but does not respond to membrane energization. It is proposed that inner membrane energization in intact cells alters the binding or environment of NPN in the outer membrane. The fluorescence recovery which occurs on anaerobiosis has two components. One component represents a reversal of the changes which occur on membrane energization. The other component of the fluorescence change is insensitive to the uncoupler CCCP and resembles the behaviour of NPN with everted membrane vesicles. It is suggested that a portion of the fluorescence events seen with NPN involves a response of the probe to changes in the inner membrane.     

Electroantennogram responses of the oriental fruit fly, Dacus dorsalis, to a spectrum of alcohol and aldehyde plant volatiles

Electroantennograms (EAGs) were recorded from unmated, laboratory-reared, male and female oriental fruit flies, Dacus dorsalis, in response to a range of between C₁ and C₁₂ carbon chain-length saturated and unaturated aliphatic alcohols and aldehydes, most all of which are known host-plant volatiles. Only two of the 35 compounds tested elicited significantly larger EAGs from female than male antennae. For the two functional-group series tested, aldehydes elicited responses greater than or equal to the responses to the alcohols. In general, the unsaturated alcohols did not elicit responses significantly different from the saturated alcohols. However, the unsaturated aldehydes, (E)-2-hexenal and 10-undecenal, elicited larger amplitude EAGs than their saturated analogs. EAGs were significantly greater for a particular carbon chain-length, with responsiveness to primary alcohols peaking at C₆ and aldehydes peaking at C₇. The (E)-2- monoenic alcohols peaked at C₆, while the (E)-3-alcohols plateaued between C₅ and C₈. The greatest EAG responses of all compounds tested were elicited by the saturated and unsaturated C₆ alcohols and aldehydes which are constitutents of the general green-leaf volatile complex that emanates from most plants. The potential adapative benefit of selective sensitivity to green-leaf volatiles is discussed in regards to foraging behaviors of oriental fruit flies.

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Résumé Des électroantennogrammes (EAG) ont enregistré les réponses, en élevages de femelles et mâles vierges de Dacus dorsalis, à une gamme de chaînes de carbones de C₁ à C₁₂ saturés et non-saturés d'alcools aliphatiques et d'aldéhydes, dont beaucoup sont connus comme substances volatiles des végétaux. Seulement 2 des 35 composés examinés ont provoqué des EAG significativement plus importants chez les femelles que chez les mâles. Pour les séries des deux groupes fonctionnels examinés, les aldéhydes ont provoqué des réponses supérieures ou égales aux alcools. En général, les réponses aux alcools nonsaturés n'étaient pas significativement différentes des réponses aux alcools saturés. Cependant, les aldéhydes non-saturés, (E)-2-hexénal et 10-undécénal, ont induit des EAG de plus grande ampleur que leurs analogues saturés. Les EAG étaient significativement les plus importants pour une chaîne de longueur particulière, la réponse aux alcools primaires culminant en C₆ et les aldéhydes en C₇. Les alcools monoéniques (E)-2- culminaient en C₆, tandis que les alcools (E)-3- étaient étales entre C₅ et C₈. Les EAG les plus importants ont été obtenus pour tous les composés examinés avec les alcools et aldéhydes en C₆ qui appartiennent à l'odeur verte complexe émise par beaucoup de plantes. Le bénéfice adaptatif potentiel de la sensibilité sélective à l'odeur verte des feuilles est examinée en fonction du comportement de prospection de D. dorsalis.

     

Effect of pupation environment on the quality of pupae and adults of the Mediterranean fruit fly

When mature larvae of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) pupated in bran rather than naked there was an increase in adult eclosion, but no difference in 9 day pupal weight, 9 day lipid content, or flight ability. There was no difference when pupation occurred at 20 or 25°C. Pupation in sand at 20°C caused severe mortality, while pupation in sand at 25°C did not. Data are presented which favour naked pupation over pupation in any medium, and evidence is presented which indicates that collection of mature larvae in a rotary tumbler rather than by the conventional popping method is economically preferable and is without significant biologically adverse effects.

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Résumé Si des larves mûres de Ceratitis capitata se nymphosent dans du son ou à l'air libre, dans le premier cas le taux d'éclosions est plus élevé, mais le poids et la teneur en lipides au neuvième jour de nymphose ainsi que l'aptitude au vol sont identiques dans les deux. Il n'y a pas de différences pour les pupaisons à 20°C et 25°C, mais la pupaison dans le sable à 20°C provoque une forte mortalité, tandis qu'à 25°C le sable n'a aucun effet. Les résultats obtenus sont favorable à une pupaison à nu plutôt que dans un substrat, et des arguments sont apportés montrant que la collecte des larves mûres dans un culbuteur rotatif ne provoque significativement pas plus d'effets nocifs que la méthode classique du saut.

     

Structure of isolated sensilla and sensory neurons in vitro: Observations on developing silkmoth antennae

Summary By combined enzymatic and mechanical treatment, it was possible to dissociate the sensory epithelium of developing antennae of male Antheraea polyphemus and A. pernyi silkmoths from the stage of separation of the antennal branches up to the early stages of cuticle deposition. Large numbers of entire developing trichoid sensilla were isolated. These are characterized by a large trichogen cell with a long apical, hair-forming process and a large nucleus. A cluster of 2–3 sensory neurons, enclosed by the thecogen cell, is situated in the basal region. The dendrites run past the nucleus of the trichogen cell into the apical process from which they protrude laterally. The nuclei of the tormogen and a 4^th enveloping cell can be distinguished near the base of the prospective hair. After further dissociation, only the neuron clusters remain, still enclosed by their thecogen cell and often attached to the antennal branch nerve via their axons. It is finally possible to disrupt the thecogen cells and the axons, leaving the sensory neurons with inner dendritic segments and axon stumps. The majority of these neurons can be expected to be olfactory.     

Surface charges of the membrane and cell adhesion substances determine the structural integrity of hair bundles from the inner ear of fish

Summary The hairs (stereocilia = stereovilli) of sensory cells from the inner ear of vertebrates are interconnected by several types of connectors, whose role is unknown. They appear to stabilize the hair bundle mechanically, and may be directly involved in mechano-electric transduction. Our transmission electron-microscopical investigation of sensory epithelia from two species of fish (Rutilus rutilus, Scardinius erythrophthalmus, both Leuciscidae) has shown that not only the connectors but also the surface charges of the membrane are important factors for determining the shape of the hair bundle and the spatial interrelation of the stereovilli. A reduction of the ionic strength in the medium leads to an increase in distance between the stereovilli. This may be the result of an extension of the spread of the surface potential of the membrane at low ionic strength. The connectors are not broken by the increase in distance between the stereovilli. They are EDTA (ethylene-diamine-tetra-acetic-acid) resistant as are some cell adhesion molecules such as N-CAM (nerve-cell adhesion molecule) and protein A from Dictyostelium discoideum. The connectors do not prevent polycation-induced fusion of adjacent stereovillar membranes.