THE CHARACTERIZATION OF HISTIDINE DECARBOXYLASE AND ITS DISTRIBUTION IN NERVES, GANGLIA AND IN SINGLE NEURONAL CELL BODIES FROM THE CNS OF APLYSIA CALIFORNICA

Histamine (HA) is present in substantial quantities in all ganglia of Aplysia californica. Within the cerebral ganglia this amine is known to be concentrated in at least two identified neurons designated C-2 neurons. In this study a combination of chemical and enzymatic analyses was employed to provide evidence for the existence of a biochemical pathway for HA synthesis in ganglia and individual neurons of this marine mollusk. Examination of extracts of individual neurons dissected from ganglia organ-cultured in the presence of [³H]histidine showed that every neuron accumulated labelled histidine, but only the HA-containing C-2 neurons synthesized and stored labelled HA suggesting that the formation of HA in Aplysia could be catalyzed by the enzyme histidine decarboxylase (HDC). HDC activity was studied with a new microradiometric assay. Many of the properties of the molluscan HDC studied were found to correspond to the vertebrate enzyme. Enzyme activity was inhibited by α-hydrazino-histidine but unaffected by concentrations of α-methyldopa or by 5-(3,4-dihydroxycinnamoyl) salicylic acid which produced nearly complete inhibition of aromatic amino acid decarboxylase activity. HDC was measurable in nervous but not other Aplysia tissues assayed. All 5 major ganglia contained HDC activity which spanned a 15-fold range between the least and most active ganglia. Only 4 of the 13 nerve trunks assayed yielded measurable enzymic activity; these active nerves were associated with the cerebral ganglia which has the highest HDC activity of all measured ganglia. Of the numerous individual neurons assayed for HDC, only the C-2 cells showed measurable enzyme activity, about 25 pmol/cell/h or 70 μmol/g protein/h. Since the activity of HDC in the HA-containing neurons was at least three orders of magnitude larger than all other neurons assayed in the cerebral and other ganglia, these data appear to provide a direct metabolic basis for the selective presence of HA in these cells, and they indicate that the cellular presence of HDC provides a useful biochemical marker for the location of HA-rich neurons in Aplysia.     

Mobilization of a Vesamicol-Insensitive Pool of Acetylcholine from a Sympathetic Ganglion by Ouabain

Abstract: These experiments investigate the release of transmitter from the perfused superior cervical ganglia of cats induced by ouabain in the absence or presence of 2-(4-phenylpiperidino)cyclohexanol (vesamicol), a blocker of acetylcholine (ACh) uptake. Ouabain, perfused through the ganglia, released ACh in a Ca²⁺-dependent way. Vesamicol caused some inhibition of the release of ACh by ouabain; however, under this condition, the Na⁺, K⁺-ATPase inhibitor released five times more transmitter than did preganglionic stimulation at 5 Hz. Also, when ganglia exposed to vesamicol were depleted of the impulse-releasable pool of ACh, subsequent perfusion with ouabain released ACh, and this included ACh newly synthesized in the presence of vesamicol; this phenomenon could be inhibited by the lack of Ca²⁺ and presence of EGTA, and was completely abolished by perfusion with a medium containing 18 mM Mg²⁺. To test whether the release of this vesamicol-insensitive Ca²⁺-dependent pool by ouabain is associated with a decrease in the number of synaptic vesicles, ganglia treated with the ATPase inhibitor after the depletion of the impulse-releasable pool of ACh were fixed for electron microscopy. In the presence of Ca²⁺, coincident with the release of the vesamicol-insensitive pool of ACh, nerve terminals were almost depleted of synaptic vesicles; ganglia treated similarly, but with medium containing 18 mM Mg²⁺ instead of Ca²⁺, were not depleted of synaptic vesicles. These results suggest that ouabain releases a vesamicol-insensitive pool of ACh from the sympathetic ganglion and also support the notion that this compartment is vesicular and its exocytosis depends on extracellular Ca²⁺. It is suggested that empty-vesicle recycling in the presence of vesamicol restricts mobilization of full vesicles to release sites.     

A putative hyperglycemic factor from the cerebral ganglia of Otala lactea (Mollusca: Pulmonata)

Mantle tissue pieces from adult Otala lactea continuously synthesized glycogen over a 72-h incubation period. Acid-saline extract of the cerebral ganglia inhibited glycogen synthesis by mantle tissue in vitro. This effect was dose-dependent. The glycogen reduction factor from the cerebral ganglia was heat stable, protease sensitive, and relatively hydrophobic. The cerebral ganglia extract also stimulated mantle glycogen phosphorylase in vitro in a dose-dependent manner. The results suggest the presence of a hyperglycemic factor in the cerebral ganglia of Otala. The molecular weight of this factor, estimated by size-exclusion chromatography, was approximately 10,000. Mammalian glucagon had no significant effect on glycogen synthesis by the mantle pieces. Accepted: 17 January 2000     

The presence and location of small cardioactive‐like peptides in larvae of Crassostrea virginica

We examined the presence and location of small cardioactive peptide (SCP)‐like neuropeptides in both the central and peripheral nervous systems of D‐hinge, newly eyed, and pediveliger larvae of Crassostrea virginica. Results indicate that SCP‐like substances are present early in development (D‐hinge larvae), and that presence in the central and peripheral nervous systems increases as the larva develops toward metamorphic competence (pediveliger larvae). In addition, in newly eyed and pediveliger larvae, SCP‐like labeling is found in a varying number of neurons within all central ganglia, with the possible exception of the accessory ganglia. Varicose labeling of axons is also documented within the ganglia, commissures, and connectives, as well as in peripheral nerves. Peripheral tissues innervated by axons exhibiting SCP‐like immunoreactivity include the velum, foot, esophagus, mantle, and various musculatures. As indicated by the location of SCP‐like labeling in various organs and tissues, it is likely that these neuropeptides modulate muscle contraction or ciliary beating in molluscan larvae such as those of C. virginica.     

Ultrastructure of the Juxtaganglionar Organ,a Putative Endocrine Gland Associated with the Cerebral Ganglia of Aplysia juliana

Summary

Electron microscopy was used to examine the morphology of a putative endocrine gland, the juxtaganglionar organ (JO), and its relation to the cerebral ganglia of the hermaphroditic opisthobranch gastropod Aplysia juliana. The JO is a well-vascularized, poorly innervated tissue of glandular cells—rich in mitochondria, lipids, ribosomes, and endoplasmic reticulum, with sparse cilia and membrane-limited secretory granules—within the connective tissue sheath just exterior to the neuronal soma in the dorsal and posterior portions of the cerebral ganglia. The cytology and organization of the JO supports its homology to the dorsal bodies of pulmonate gastropods, which axe endocrine organs known to release one or more female gonadotropic factors.     

A comparative study of leucokinin-immunoreactive neurons in insects

Antisera were raised against leucokinin IV, a member of the leucokinin peptide family. Immunohistochemical localization of leucokinin immunoreactivity in the brain of the cockroach Nauphoeta cinerea revealed neurosecretory cells in the pars intercerebralis and pars lateralis, several bilateral pairs of interneurons in the protocerebrum, and a group of interneurons in the optic lobe. Several immunoreactive interneurons were found in the thoracic ganglia, while the abdominal ganglia contained prominent immunoreactive neurosecretory cells, which projected to the lateral cardiac nerve. The presence of leucokinins in the abdominal nerve cord was confirmed by HPLC combined with ELISA. Leucokinin-immunoreactive neurosecretory cells were also found in the pars intercerebralis of the cricket Acheta domesticus and the mosquito Aedes aegypti, but not in the locust Schistocerca americana or the honey bee Apis mellifera. However, all these species have leucokinin-immunoreactive neurosecretory cells in the abdominal ganglia. The neurohemal organs innervated by abdominal leucokinin-immunoreactive cells were different in each species.     

β2-Adrenoreceptor immunoreactivity in cardiac ganglia of the guinea pig

Summary Previous pharmacological studies in co-culture systems have indicated, the presence of β-adrenoreceptors on intrinsic cardiac neurons of the guinea pig (Horackovaet al., 1993) but radiologand binding studies of tissue sections failed to provide a definite answer as to the presence of such receptors on cardiac neuronsin situ, due to the iodine-binding properties of cardiac nerve bundles and ganglia (Molenaaret al., 1992). We therefore addressed this question by immunohistochemistry, using antisera raised against synthetic peptides of the β2-adrenoreceptor. For comparison, cholinergic and catecholaminergic neurons were identified immunohistochemically by means of antibodies against the enzymes involved in the synthesis of acetylcholine (choline acetyltransferase), and of catecholamines (tyrosine hydroxylase). Virtually all intrinsic cardiac neurons contained both β2-adrenoreceptor- and choline acetyltransferase-immunoreactivities. In addition, some nerve fibre bundles exhibited β2-adrenoreceptor-immunoreactivity. Several ganglia were innervated by tyrosine hydroxylase-immunoreactive axons, but the majority of ganglia did not receive tyrosine hydroxylase-immunoreactive nerve terminals, and additional intraganglionic sources of catecholamine synthesis could not be identified. Thus, the results are in favour of β-adrenergic modulation of guinea pig cardiac ganglia by humorally and, partially, by locally released catecholamines.     

SEROTONIN AND FREE AMINO ACID ANALYSIS OF GANGLIA AND ISOLATED NEURONES OF APLYSIA DACTYLOMELA

—The presence of serotonin and different amino acids was investigated in the ganglia and in isolated giant neurones of Aplysia dactylomela. With a few exceptions the pattern of substances was similar in all the ganglia. Of the many identified neurones studied only one giant neurone located in each cerebral ganglion was found to contain serotonin. GABA was detected in most extracts, including those of the serotonin-containing neurone, known cholinergic, and known neurosecretory neurones. Putrescine, recently detected in extracts of nervous tissue and isolated neurones of Helix, was not detected in Aplysia nervous tissue.     

Effect of neighbour presence and soil volume on the growth of Andropogon gerardii Vitman

Background: Theory predicts that plants can reduce their fitness in the presence of neighbours by allocating resources to root growth, in order to pre-empt resource capture. A number of studies that have tested this idea have done so by using experiments where neighbour presence is confounded with soil volume.

Aims : To avoid confounding effects of neighbour presence and soil volume we adjusted these variables independently from one another.

Methods: We grew Andropogon gerardii with and without neighbours, holding soil volume available to each plant constant, and compared plant performance with a treatment where both neighbour presence and soil volume were varied. We also grew plants with a quarter of the soil volume but four times the nutrient concentration to determine if changes in plant growth in response to soil volume are caused by access different levels of soil resources.

Results: We found no evidence that plants adjust root growth to the presence of neighbour roots alone. We did, however, find a significant reduction in plant growth when soil volume was reduced. The reduction was overcome by increasing nutrient concentrations in the growth media.

Conclusions: Our results suggest the effects of soil volume on plant growth are mainly due to changes in nutrient availability.     

<Emphasis Type="Italic">Synechocystis</Emphasis> ferredoxin-NADP<Superscript>+</Superscript> oxidoreductase is capable of functioning as ferric reductase and of driving the Fenton reaction in the absence or presence of free flavin

We purified free flavin-independent NADPH oxidoreductase from Synechocystis sp. PCC6803 based on NADPH oxidation activity elicited during reduction of t-butyl hydroperoxide in the presence of Fe(III)-EDTA. The N-terminal sequencing of the purified enzyme revealed it to be ferredoxin-NADP⁺ oxidoreductase (FNR _S ). The purified enzyme reacted with cytochrome c, ferricyanide and 2,6-dichloroindophenol (DCIP). The substrate specificity of the enzyme was similar to the known FNR. DNA degradation occurring in the presence of NADPH, Fe(III)-EDTA and hydrogen peroxide was potently enhanced by the purified enzyme, indicating that Synechocystis FNR _S may drive the Fenton reaction. The Fenton reaction by Synechocystis FNR _S in the presence of natural chelate iron compounds tended to be considerably lower than that in the presence of synthetic chelate iron compounds. The Synechocystis FNR _S is considered to reduce ferric iron to ferrous iron when it evokes the Fenton reaction. Although Synechocystis FNR _S was able to reduce iron compounds in the absence of free flavin, the ferric reduction by the enzyme was enhanced by the addition of free flavin. The enhancement was detected not only in the presence of natural chelate iron compounds but also synthetic chelate iron compounds.     

Characterization of the tyraminergic system in the central nervous system of the locust,Locusta migratoria migratoides

Tyramine occurs in the central nervous system (CNS) of the migratory locust,Locusta migratoria migratoides. The distribution of tyramine within the CNS does not parallel that of octopamine. Tyramine is synthesised from tyrosine in the presence of tyrosine decarboxylase. A second decarboxylase in the CNS is active against 5HTP and DOPA. The locust ganglia incorporate tyramine by high- and low-affinity uptake processes that appear to be independent of dopamine and octopamine. Depolarisation of the locust ganglia by high potassium concentration results in calcium-dependent release of incorporated [³H]tyramine.     

The induction of valine dehydrogenase activity from Streptomyces by L-valine is not repressed by ammonium

Summary Activity of valine dehydrogenases (VDH) from Streptomyces aureofaciens and S. fradiae is strongly induced by L-valine even in the presence of 25mM NH₄ ⁺. When added into 16 h-old cultures growing with 100mM NH₄ ⁺, L-valine induced the synthesis of VDH. The results indicate that Streptomyces can utilize L-valine in the presence of NH₄ ⁺, and the induction of VDH activity by L-valine is not repressed by NH₄ ⁺.     

Diet composition influences annual breeding success of Montagu's Harriers Circus pygargus feeding on diverse prey

Capsule Three similarly sized passerine species with various breeding strategies behaved differently in the presence of models of mammalian and avian predators.

Aims We tested whether solitary breeders behave in the presence of a predator in order to avoid disclosing their nest site in: hole-nesting Pied Flycatcher Ficedula hypoleuca, shrub-nesting Blackcap Sylvia atricapilla and ground-nesting Meadow Pipit Anthus pratensis.

Methods The behaviour of parents in response to two types of predator model (visually oriented Corvid birds and olfactory oriented Mustelid mammals) placed sequentially at 40, 5 and 1 m distance from the nest was recorded from a shelter.

Results The hypothesis was supported in open-nesting species (Meadow Pipit and Blackcap), as parents did not approach the nest in the presence of a predator. In the hole-nesting Pied Flycatcher the parents disclosed the nest site in most cases (by entering the nest). The intensity of alarm calling increased with decreasing distance of a predator from the nest in all species except Meadow Pipit in the presence of Raven Corvus corax models. The intensity of attacking changed only in Meadow Pipit with decreasing distance of Stoat Mustela erminea from the nest.

Conclusion The results showed that anti-predator behaviour was species-specific, depending on type of predator, habitat and nest inaccessibility.     

Crop presence,but not genetic diversity,impacts on the rare arable plant Valerianella rimosa

Background: Intensive farming affects farmland biodiversity, and some arable plants in particular. Increasing crop genetic diversity can increase crop productivity or resilience and could also benefit rare arable plants.

Aims: We examined whether barley presence, sowing density and genetic diversity impacted the rare plant Valerianella rimosa and explored possible underlying mechanisms.

Methods: In a field study near Dundee, Scotland, we sowed plots of five single barley genotypes, and all five genotypes combined, at three densities; we also had barley-free plots. Valerianella seeds were sown into half of all plots. Measured responses included early-season cover and harvest biomass of barley and common weeds, abiotic parameters (soil moisture, light) and establishment, biomass and seed production by V. rimosa.

Results: Barley presence promoted V. rimosa establishment early in the growing season, but without barley density or genetic diversity effects. By harvest, the impact of barley presence on V. rimosa abundance was lost; there were no effects on Valerianella seed production. Barley negatively impacted common weeds, but V. rimosa did not benefit from any indirect facilitation by barley, being bigger without barley.

Conclusions: Early beneficial effects of barley on V. rimosa abundance appear offset by late-season competition. However, limited impacts of barley on V. rimosa reproductive success, and negative impacts on common weeds, indicate crops might play a role in conservation management of rare arable plants by creating space in the weed community.     

Decrease in the number of high-affinity opiate binding sites during the aging process inMytilus edulis (Bivalvia)

As Mytilus edulis(Bivalvia) ages, the number of stereospecific opiate binding sites in the visceral ganglia significantly decreases. The number of high-affinity etorphine sites decreased by 30%; the number of naloxone sites decreased by 23%. The affinity of the ligands did not vary with age. In addition, the augmentation by opioids of dopamine levels in the visceral ganglia was less in older animals and was accompanied by a reduced sensitivity of the ganglia to etorphine and DAMA (d-Ala ²,Met⁵-enkephalinamide).     

Norlaudanosoline and Nicotine Increase Endogenous Ganglionic Morphine Levels: Nicotine Addiction

Summary 1. Given the presence of morphine, its metabolites and precursors, e.g., norlaudanosoline, in mammalian and invertebrate tissues, it became important to determine if exposing normal excised ganglia to norlaudanosoline would result in increasing endogenous morphine levels.2. Mytilus edulis pedal ganglia contain 2.2 ± 0.41 ng/g wet weight morphine as determined by high pressure liquid chromatography coupled to electrochemical detection and radioimmunoassay.3. Incubation of M. edulis pedal ganglia with norlaudanosoline, a morphine precursor, resulted in a concentration- and time-dependent statistical increase in endogenous morphine levels (6.9 ± 1.24 ng/g).4. Injection of animals with nicotine also increased endogenous morphine levels in a manner that was antagonized by atropine, suggesting that nicotine addiction may be related to altering endogenous morphine levels in mammals.5. We surmise that norlaudanosoline is being converted to morphine, demonstrating that invertebrate neural tissue can synthesize morphine.     

Hydroxylation of steroids by Fusarium oxysporum, Exophiala jeanselmei and Ceratocystis paradoxa

The potential of Fusarium oxysporum var. cubense UAMH 9013 to perform steroid biotransformations was reinvestigated using single phase and pulse feed conditions. The following natural steroids served as substrates: dehydroepiandrosterone (1), pregnenolone (2), testosterone (3), progesterone (4), cortisone (5), prednisone (6), estrone (7) and sarsasapogenin (8). The results showed the possible presence of C-7 and C-15 hydroxylase enzymes. This hypothesis was explored using three synthetic androstanes: androstane-3,17-dione (9), androsta-4,6-diene-3,17-dione (10) and 3α,5α-cycloandrost-6-en-17-one (11). These fermentations of non-natural steroids showed that C-7 hydroxylation was as a result of that position being allylic. The evidence also pointed towards the presence of a C-15 hydroxylase enzyme.The eleven steroids were also fed to Exophialajeanselmei var. lecanii-corni UAMH 8783. The results showed that the fungus appears to have very active 5α and 14α-hydroxylase enzymes, and is also capable of carrying out allylic oxidations.Ceratocystis paradoxa UAMH 8784 was grown in the presence of the above-mentioned steroids. The results showed that monooxygenases which effect allylic hydroxylation and Baeyer–Villiger rearrangement were active. However, redox reactions predominated.     

Demonstration by organ culture of a cerebral hormone stimulating the secretion of testicular androgen in the oligochaete annelid,Eisenia foetida f. typica Sav.

Previous culture experiments revealed that, in reproductively active E. foetida, the central nervous sytem (CNS) produces a neurohormone necessary for the secretion of an androgen by testes. The tracing of the origin of this neurohormone has now been done by culturing in vitro ovaries and testes with different ganglia of the nerve cord. In the presence of cerebral ganglia (CG), the percentage of testes transformed into ovotestes was low (3%), while reaching respectively 32% and 17% in the associations with sub-esophageal ganglia (SEG) or ganglia of the male or female genital segments (GS ♂ or ♀). Only the neurohormone produced by CG was able to stimulate secretion of the testicular androgen, the existence of which was proved by previous experiments. The secretion of this androgen was enough to transform 28% of the ovaries into ovotestes. On the other hand, no masculinization of the ovaries was observed in the presence of various ganglia of the ventral nerve cord (SEG, GS ♂ or ♀). It appears, therefore, that the secretion of the testicular androgen is stimulated by the CG.     

Sodalis glossinidius presence in wild tsetse is only associated with presence of trypanosomes in complex interactions with other tsetse-specific factors

Background

Susceptibility of tsetse flies (Glossina spp.) to trypanosomes of both humans and animals has been associated with the presence of the endosymbiont Sodalis glossinidius. However, intrinsic biological characteristics of the flies and environmental factors can influence the presence of both S. glossinidius and the parasites. It thus remains unclear whether it is the S. glossinidius or other attributes of the flies that explains the apparent association. The objective of this study was to test whether the presence of Trypanosoma vivax, T. congolense and T. brucei are related to the presence of S. glossinidius in tsetse flies when other factors are accounted for: geographic location, species of Glossina, sex or age of the host flies.

Results

Flies (n = 1090) were trapped from four sites in the Shimba Hills and Nguruman regions in Kenya. Sex and species of tsetse (G. austeni, G. brevipalpis, G. longipennis and G. pallidipes) were determined based on external morphological characters and age was estimated by a wing fray score method. The presence of trypanosomes and S. glossinidius was detected using PCR targeting the internal transcribed spacer region 1 and the haemolysin gene, respectively. Sequencing was used to confirm species identification. Generalised Linear Models (GLMs) and Multiple Correspondence Analysis (MCA) were applied to investigate multivariable associations. The overall prevalence of trypanosomes was 42.1%, but GLMs revealed complex patterns of associations: the presence of S. glossinidius was associated with trypanosome presence but only in interactions with other factors and only in some species of trypanosomes. The strongest association was found for T. congolense, and no association was found for T. vivax. The MCA also suggested only a weak association between the presence of trypanosomes and S. glossinidius. Trypanosome-positive status showed strong associations with sex and age while S. glossinidius-positive status showed a strong association with geographic location and species of fly.

Conclusions

We suggest that previous conclusions about the presence of endosymbionts increasing probability of trypanosome presence in tsetse flies may have been confounded by other factors, such as community composition of the tsetse flies and the specific trypanosomes found in different regions.

     

Synthesis and characterization of poly-O-methyl-[n]-polyurethane from a d-glucamine-based monomer

Aminoalditol 1-amino-1-deoxy-d-sorbitol (1) was readily converted into 2,3,4,5-tetra-O-methyl derivative 5, a key precursor of a sugar-based [n]-polyurethane. For the polymerization, the free amino or primary hydroxyl groups of 5 were selectively activated and employed as starting monomers in two alternative procedures. Thus, the amino function of 5 was converted into the isocyanate derivative by treatment with di-tert-butyltricarbonate, and polymerized in situ in the presence of Zr(IV) acetylacetonate. The resulting poly(1-amino-1-deoxy-2,3,4,5-tetra-O-methyl-d-sorbitol)urethane (8) had a moderate molecular weight and showed the presence of urea units. The alternative synthesis of 8 involved the activation of the free hydroxyl group of 5 as the corresponding phenylcarbonate. The polymerization of this α-amino-ω-phenylcarbonate alditol monomer does not require a metal catalyst. The resulting material exhibited an improved molecular weight and higher purity than that obtained via the isocyanate. [n]-polyurethane 8 was highly soluble in water as well as in common organic solvents (chloroform, acetone, ethyl acetate, etc) and was obtained as an amorphous material which was characterized thermally and spectroscopically.